BACKGROUND
genomic mapping of histone modifications, their writers, readers and erasers as well as transcription factors  has become a house-hold approach to study the genome-wide regulation of gene expression programs  <cit> .
the most widely applied method to generate such global mapping data is chromatin immunoprecipitation coupled with high-throughput sequencing , which however generally requires millions of cells as input material .
scarcity of cells in distinct, isolated in vivo populations such as phenotypically defined hematopoietic stem and progenitor cells has thus hampered direct experimentation on these.
examining such sub-populations is of intense interest for the elucidation of mechanisms governing lineage choice and commitment as well as transcriptional de-regulation in disease.
some researchers have cultured harvested cells to achieve sufficient cell numbers and performed chip-seq on cells undergoing differentiation in vitro, but this approach can give rise to biases due to culture conditions .
recent advances in the methodology have demonstrated successful chip-seq on very limited cell numbers  <cit> .
however, these techniques are impeded by additional rounds of pre-amplification, potentially making them prone to introduce artifacts.
one of these methods was established on cultured cells  <cit> , while the other is limited to showing chip-seq with antibodies against histone modifications  <cit> , yielding significantly more immunoprecipitated dna than chip with antibodies to transcription factors.
furthermore, the complexity and cost of pre-amplification deters the implementation of the existing methods in many laboratories.
another recent method uses an elegant step of pre-chip indexing of histone  <dig> containing chromatin fragments, allowing downstream distinction of input material  <cit> .
the mixed inputs may thus function as mutual carriers during single-tube, small cell number chip.
importantly, the indexing step selects against nucleosome-poor genomic regions, making this approach less useful for unbiased investigation of genome-wide tf occupancy.
here, we describe a straightforward and versatile work-flow for both transcription factor and histone mark chip-seq on low abundance cell populations isolated directly from the in vivo setting.
a key element is the introduction of bacterial carrier dna at the amplification step.
this eliminates the previous need for pre-amplification and makes possible robust generation of sequencing libraries from picogram amounts of chip dna.
RESULTS
histone mark chip-seq of hematopoietic cell populations
the scarcity of biologically relevant in vivo material is often barring global level investigations into normal development as well as the aberrant regulation behind cancer and other complex diseases.
of particular interest are the genome-wide binding patterns of transcription factors and the associated epigenetic profiles, which may pinpoint aberrant molecular mechanisms underlying transcriptional dysregulation and development of disease.
here, we use a standard facs regimen  to isolate a specific hematopoietic gmp-blast population from cebpap30/p <dig> mice expressing a truncated variant of the myeloid transcription factor cebpa  <cit> .
these mice develop acute myeloid leukemia with complete penetrance, and have been studied in detail  <cit> .
however, the precise molecular dysregulation driving leukemogenesis remains obscure.
we therefore developed a chip-seq assay compatible with the numbers of isolated leukemic cells from the in vivo context.
first, we optimized our chip protocol for small cell numbers, which is described in detail here for clarity.
immediately after the sorting procedure, isolated cells were exposed to formaldehyde for cross-linking chromatin-associated proteins to the dna, washed and snap frozen in liquid nitrogen.
next, they were subjected to sonication to break the chromatin into suitably sized fragments .
we found that careful inspection of the dna size distribution of each batch of chromatin was useful to prevent further processing of low quality samples.
this was achieved either by processing a parallel sample of c-kit enriched bm cells, providing a sufficient cell number for standard gel electrophoresis, or by direct inspection of each sample using the bioanalyzer dna <dig> assay ).
chromatin from roughly  <dig>  cells, equivalent to 250–300 ng of naked dna, was used as input for each chip experiment with antibodies against the histone marks h <dig> lys <dig> trimethylation  or h <dig> lys <dig> trimethylation , performed in siliconized tubes with optimized washing conditions and titrated antibody and antibody-binding beads .
utilizing a thorough approach of extended protein degradation and de-crosslinking steps, as well as phenol-chloroform extraction for retrieving chip dna ensured robust high recovery.
this approach allowed us to effectively enrich for genomic sequences associated with either h3k27me <dig> or h3k4me <dig> as assessed by quantitative pcr  .
the h3k27me <dig> chip produced ca.
2 ng of dna for each sample.
by making minor but important changes to the standard illumina protocol, we were able to consistently amplify the 2 ng chip dna to generate libraries for high-throughput sequencing .
the h3k4me <dig> chip yielded an amount of dna below the effective range of standard absorbance or fluorescence assays.
we circumvented this obstacle by taking advantage of the fluorescence nanodrop instrument, which allows reliable detection of dna down to 5 pg/ul in a  <dig> ul sample volume .
with this approach, h3k4me <dig> chip dna was measured to ca.
700 pg dna, which we pooled to obtain the 2 ng sufficient for robust amplification .
using the illumina hiseq platform, we deep sequenced two libraries derived from two biologically independent samples for each of the two histone marks .
we processed the aligned reads into genomic coverage profiles using standard procedures .
visual analysis of the profiles suggested a good concordance with previous findings  <cit> , showing enrichment of the h3k27me <dig> mark in gene bodies, intergenic regions as well as promoters and h3k4me <dig> in gene promoter regions .
a quantitative analysis mapped h3k27me <dig> reads as 6% in promoter  and 56% in gene body locations , while 21% of h3k4me <dig> reads resided in promoters .
promoter h3k4me <dig> modifications were positively and h3k27me <dig> negatively correlated with activity of associated genes, as observed previously  .
finally, we assessed the reproducibility of our chip-seq approach by comparing coverage in promoter regions from two biologically independent replicates and found a high degree of correspondence, both by visual inspection  and quantitative measures .
in the examined cell type, we observe a near mutually exclusive pattern of h3k4me <dig> and h3k27me <dig> marks in promoters , as opposed to e.g.
embryonic stem cells displaying a set of double-marked promoters at poised genes, a hall-mark of the undifferentiated state  <cit> .figure  <dig>
generation of genomic coverage maps of dna-associated proteins from rare, isolated cell populations.
schematic depiction of small-scale chip-seq workflow, including pico-gram dna quantification and addition of fragmented bacterial dna carrier.
chip’ed dna in black, carrier-dna in light grey.
validation of small-scale chip-seq histone mark maps.
two h3k27me <dig> and h3k4me <dig> profiles and refseq gene positions across a 400 kb region on chromosome  <dig>   genomic distribution of all mapped reads, rounded numbers.
scatterplots of promoter histone methylation and mrna expression in gmp-phenotype blast cells.
each data point represents a single gene.
quantile normalized.
quantitative comparisons of promoter read coverage  from two independent biological replicates of each histone modification using 1-kb tss centered bins.
quantile normalized, 1/ <dig> diagonal indicated by dashed line .
seqminer cluster heatmaps showing signal intensities for both replicates of h3k27me <dig> and h3k4me <dig> in a 20000 bp region centered on refseq tss positions.
transcription factor chip-seq of specific, isolated cell populations
chromatin-associated proteins with limited genomic distributions generally yield less chip dna when compared to chromatin components showing broader distributions.
this is in line with our observations for h3k4me <dig> versus h3k27me <dig> modifications, with characteristic peak-like and broad distributions, respectively .
accordingly, we expected transcription factor chip to yield even less output dna.
to address this issue we used a larger input cell number.
we performed chip with antibodies against cebpa, a transcription factor known to be expressed in the gmp blast population  <cit> , using chromatin from  <dig>  or  <dig>  cells, corresponding to 500–1000 ng input dna per sample.
by performing qpcr for amplicons covering known cebpa target sequences, we verified the quality of the immuno-precipitation step .
utilizing the fluorescent nanodrop, we measured the chip dna yields of down to 250 pg , an amount prohibitive for standard use as a starting point for illumina amplification.
we reasoned that an absolute loss during the amplification procedure could be ameliorated by adding a dna carrier.
first, we set out to test the use of a synthetic 120-mer oligo devoid of the terminal 3’-oh group, which would prevent ligation to the amplification adaptors and hence preclude amplification during the pcr step.
this gave rise to unacceptable biases as displayed by non-linear amplification of cebpa enriched regions , probably due to low complexity of the dna pool during pcr amplification.
we next hypothesized that addition of complex carrier dna that could be amplified would be ideally suited to surpass this problem.
hence, we performed an in silico mappability test of  <dig> million 50-bp randomly extracted e. coli sequences, of which  <dig> mapped to the mouse genome and  <dig>  to the human counterpart .
adding fragmented bacterial dna  to the cebpa chip dna  produced the 2 ng established to perform robustly in amplification.
by deep sequencing the resulting compound library, we were able to map up to 20% of the obtained sequences to the mouse genome .
this corresponded with the ratio between chip dna and bacterial carrier and typical chip-seq mapping frequencies.
cebpa peaks were observed in promoter proximal positions characteristic of many transcription factors  .
quantitative assessment of cebpa peak positions revealed a genomic distribution analogous to a thoroughly validated cebpa chip-seq dataset obtained with liver cells   <cit> .
further visual inspection identified several examples of cebpa peaks shared between hepatocyte and myeloid progenitor data sets .
strikingly, the two archetypical homeostatic liver genes  and carbamoyl-phosphate synthase  <dig> ) displayed cebpa peaks in hepatocytes, but not in myeloid cells.
conversely, many genes characteristic of the myeloid lineage displayed cebpa peaks only in the myeloid chip-seq dataset , colony stimulating factor  <dig> receptor , matrix metallopeptidase  <dig> , selectin l, colony stimulating factor  <dig> , fc fragment of igg, low affinity iiib, receptor ) .
several of these are known cebpa targets.
two additional pieces of evidence supported the validity and specificity of our cebpa genomic occupancy data.
firstly, the top hit of a de novo motif search in the enriched sequences matched the known cebpa binding logo , and this motif was found strongly enriched in the peak centers .
secondly, robust conservation in these sequences were centered on cebpa motifs, implying functional evolutionary constrain .
visual and quantitative comparisons of coverage from two biologically independent repeats indicated a high degree of reproducibility .
collectively, these data provide evidence that transcription factor chip-seq on small-cell-number populations is possible using bacterial dna as a carrier during the amplification step.figure  <dig>
validation of small-scale transcription factor chip-seq.
profiles generated from  <dig> pg cebpa chip-dna.
region spanning a  <dig> kb region on chromosome  <dig>   genomic distribution of top  <dig>  cebpa peaks  from gmp-blasts compared with  <dig>  cebpa peak positions in hepatocytes , rounded numbers.
cebpa coverage tracks from gmp-blast and hepatocyte gene loci of shared, specific liver or myeloid cell expression.
see text for full gene names.
de-novo motif search output from meme , and top match jaspar motif cebpa , both in logo format .
data set as in d.  distribution of the top match motif cebpa  in  <dig> bp regions centered on cebpa- <dig> peak summits.
usf <dig>  included for comparison.
phylo-p conservation plots in  <dig> bp  and  <dig> bp  regions centered on cebpa-motifs in cebpa- <dig> peaks.
green lines delineate motif position.
quantitative comparison of mean read coverage of two chip-seq repeats, cebpa- <dig> and cebpa- <dig>  using cebpa- <dig> peak regions defined by macs <dig>  quantile normalized, 1/ <dig> diagonal indicated by dashed line .
macs <dig> peak boundaries for cebpa- <dig>  seqminer cluster heatmaps showing signal intensities for cebpa- <dig> and cebpa- <dig> profiles in  <dig> bp regions centered on peak summits.
for f and g, peak set as in b.
amplification from picogram amounts of chip dna
many immunophenotypically defined hematopoietic compartments, e.g.
the hematopoietic stem cells, consist of a very limited number of cells.
chip from small cell populations, as has been done previously by several groups  <cit> , consistently yield very limited quantities of chip dna.
hence, we wanted to investigate if our carrier dna amplification approach could be applied on pico-gram-scale amounts of dna.
to test this, we generated a series of compound dna illumina amplified libraries with varying ratios of bacterial carrier dna and chip dna from three pooled cebpa chip samples  to allow direct comparisons between libraries.
aiming to minimize the dilution ratio, some of these libraries were generated using total input amounts of  <dig> or 500 pg.
the resulting four libraries , all displayed amplification output yields and size distributions comparable to libraries generated from 2 ng dna .
high-throughput sequencing of these libraries resulted in mapping frequencies close to the expected based on standard chip-seq mapping efficiency and ratios of chip dna to bacterial carrier dna.
e.g.
from a read number of roughly  <dig> million for cebpa- <dig>   <dig>  million mapped uniquely to the mouse genome .
visually, genomic coverage profiles for each of the four new libraries closely matched our previous cebpa tracks .
an analysis of the degree of correlation between dilute libraries and cebpa- <dig> indicated consistent, high reproducibility .
strikingly, the library containing least chip dna  display a pearson correlation of  <dig>  with the 300 pg cebpa- <dig> library and overall recapitulate the genomic coverage of this library .figure  <dig>
comparison of pico-scale transcription factor libraries.
cebpa profiles of a  <dig> kb region on chromosome  <dig>  based on progressively lower amounts of input chip-dna and bacterial carrier .
quantitative comparisons of mean read coverage of cebpa- <dig> versus cebpa- <dig> /-4/-5/- <dig>  chip-seq profiles in cebpa- <dig> peak regions defined by macs <dig>  quantile normalized, 1/ <dig> diagonal indicated by dashed line .
seqminer cluster heatmaps showing signal intensities for profiles cebpa- <dig> and cebpa- <dig> at  <dig> bp regions centered on peak summits.
for b and c, peak set as in figure 3b.
next, we investigated the diluted libraries for presence of cebpa peaks at known cebpa targets , alpha and - beta, prostaglandin-endoperoxide synthase  <dig> and interleukin  <dig> receptor, alpha), and found these in all coverage profiles   <cit> .
to test our panel of libraries further, we performed qpcr to quantify amplicons corresponding to the cebpa enriched regions mentioned above, which indicated reproducible and practically uniform amplification across dilution ratios and input amounts .figure  <dig>
test of pico-scale chip-seq amplification linearity.
cebpa profiles of promoter regions of six known cebp targets.
qpcr validation of linearity.
amplicons positioned in cebpa peak regions from a. chip-dna  and bacterial carrier dna  amounts indicated.
chip-seq using  <dig>  cells
to investigate if our methodology allows chip-seq on limited cell numbers, we transplanted a new cohort of mice with the leukemic strain also used in the above and facs-isolated batches of  <dig>  gmp-blasts.
we optimized the chromatin preparation procedure to ensure maximal input material for the small-cell-number chip .
thorough optimization of chip conditions produced robust enrichments for histone mark  as well as tf  chip, both of which compared favorably to chips done according to a previously published  <dig>  cell chip methodology by zwart et al.
<cit>  .
the major advance in the previous study was addition of a combined mrna/histone carrier/blocker during the chip step.
we amended our protocol to include such a carrier, resulting in even better enrichment as assessed by qpcr .
a small amount of  <dig>  cell chip-dna was used for examining enrichment and establishing quantity, leaving ca.
60 pg dna.
parallel generation of sequencing libraries with and without bacterial carrier demonstrated that at the 60 pg chip-dna range, carrier is required for robust production of high-quality illumina libraries .
both visual inspection and quantitative analyses of genomic coverage tracks from  <dig>  cell chips revealed good correspondence with libraries generated from biologically independent higher-cell-number chips .
cebpa- <dig> peaks were to a high degree shared with a biological repeat , the diluted library  and the low-cell-number chip  .
importantly, the h3k4me3-10k profile generated from 60 pg chip-dna, which display substantially fewer mapped reads , is highly similar to the h3k4me3- <dig> profile from 2 ng dna resulting from chip without using bacterial carrier for amplification .figure  <dig>
<dig>  cell histone mark and transcription factor chip-seq.
chip using  <dig>  sorted gmp-blasts as input.
our protocol  with and without mrna/histone carrier, compared to the zwart protocol.
qpcr assessment of enrichment ratios, n = 4- <dig>  error bars show sd.
*** = p <  <dig> , t-test, holm-sidak correction for multiple testing.
h3k4me3-chip, ratios are amplicon smc <dig> versus the negative sfi <dig>   cebpa-chip, ratios are amplicon ptgs <dig> versus the negative sfi <dig>   h3k4me <dig> and cebpa chip-seq profiles across a region spanning a 130 kb region on chromosome  <dig>  using  <dig>   or  <dig>   cells as input  and cebpa chip-seq profiles of using  <dig>   or  <dig>   cells as input .
quantitative comparisons of mean read coverage of h3k4me3- <dig> versus h3k4me3-10 k  and cebpa- <dig> versus cebpa-10 k .
all settings as described above.
peak overlaps between alpha- <dig> and alpha- <dig>  cebpa- <dig> and cebpa-10 k. macs <dig> peaks were ranked for fdr  and the cebpa- <dig> top 1% set tested against peaks scoring fdr < 10− <dig>  in the fdr top 1% or in top  <dig> .
schematic bar diagram depicting summed ngs-qc  score for each of the listed small-scale chip-seq data sets.
listed is number of cells used as input material, source of material  and antibody target .
the chip-dna fraction column denotes amplification of a fraction of dna from a higher-cell-number chip corresponding to the indicated number of cells.
the carrier column indicates use of mrna/histone as carrier during the chip procedure .
all scores represent best replicate.
ngs-qc sum score is calculated as a sum of quartile scores  for read sub-sampling of  <dig>   <dig> and 50% of total mapped reads.
the recent establishment of the online next generation sequencing quality control  generator, a useful cross-platform quality assessment tool  <cit> , allowed a quantitative comparison of our chip-seq approach with already published methods .
our methodology robustly produced ngs-qc scores matching or exceeding those from previous approaches .
specifically, shankaranarayanan  amplification  <cit>  resulted in considerably lower ngs-qc scores at the  <dig>  cell range, while adli amplification  <cit>  and the zwart methodology  <cit>  produced data sets with comparable scores for histone modification or transcription factor chip-seq profiles, respectively .
discussion and
CONCLUSIONS
here, we present a complete work-flow for chip-seq on the limited cell populations of distinct in vivo cell compartments.
by isolating a defined cell population with a standard immunophenotypic sorting regimen and using a rigorous and reliable chip approach, we were able to enrich regions marked by both broadly  and more narrowly  distributed histone modifications as well as the precise regions defined by occupancy of the central hematopoietic transcription factor, cebpa.
importantly, the fluorescence nanodrop instrument allowed us to reliably measure the obtained picogram amounts of chip dna.
we demonstrate that these amounts of chip dna could be faithfully amplified to generate libraries for illumina sequencing by addition of fragmented e. coli carrier dna.
further, we show the resulting coverage profiles to match previous data for the actively transcribed gene associated  and repressed gene associated  histone marks, and to specifically recover known myeloid specific cebpa target regions.
we provide evidence that cebpa bound regions from a pool of chip’ed dna down to 50 pg are amplified in a linear manner with our technique.
finally, we demonstrate that our straight-forward methodology can produce high-quality chip-seq coverage tracks from as little as  <dig>  isolated in vivo cells using antibodies against histone mark  as well as transcriptional factor  antibodies.
importantly, the h3k4me3-chip  <dig>  cell genomic coverage profile is very similar to the profiles generated without bacterial carrier , indicating that the addition of bacterial carrier does not impede amplification or illumina sequencing.
our methodology can be used to elucidate important biological circuitries at a global level.
for instance, we can clearly detect differences of direct targets of cebpa in specific cell types as illustrated in figure  <dig>  importantly, two recently published studies demonstrate the usefulness of our approach by revealing molecular mechanisms behind initiation of acute myeloid leukemia and regulation of hematopoietic stem cells  <cit> .
furthermore, our technology should combine easily with other genomics approaches, for instance chia-pet  <cit> , to permit generation of libraries from otherwise prohibitively small amounts of dna.
other available methodologies that allow generation of chip-seq data from limited amounts of input material rely on an extra amplification step, based either on custom designed random primers or t <dig> rna-polymerase technology  <cit> .
even as these methods are very useful, introducing extra and complex steps in the amplification procedure will inevitably increase the risk of errors, and is costly and time-consuming.
our method is based on the conceptually simple addition of non-mapping dna as carrier and should as such be easy to implement in any laboratory that already performs illumina platform chip-seq.
a disadvantage of our approach is the added cost of sequencing e. coli dna, generating data that will be discarded.
this drawback increases with the ratio between bacterial and chip dna, i.e.
as fewer cells are used or less material recovered, for example as a result of poor antibodies or low expression of the chiped factor.
we have tried to address this issue by demonstrating the feasibility of using just  <dig> picogram total input material for the amplification procedure.
the steady decline of sequencing prices should also help reduce this disadvantage.
several groups have optimized chip from very small cell numbers , whereas our study focusses on refining the amplification step of chip-seq.
some reports have shown the addition of carrier chromatin during the chip stage to facilitate the application on small cell numbers, but these methodologies are either not tested or incompatible with high-throughput sequencing  <cit> .
recently, zwart et al.
demonstrated an increase in the chip-seq signal to noise ratio by adding carrier consisting of rna and histones  <cit> .
while chip protocols generally include bovine serum albumin as a carrier , zwart and coworkers speculate that combined oligonucleotide rna and histones mimic chromatin better, and hence offer improved blocking of spurious binding.
both components are degraded prior to the amplification step, making the modification suitable for use in chip-seq.
we compare a low-cell-number optimized version of our methodology and find it to surpass the zwart approach for the tested antibodies .
nevertheless, adopting the mrna/histone chip level carrier considerably improves our protocol , clearly demonstrating the value of the zwart at al.
contribution to the development of chip technology.
in summary, our study, in combination with the progress of sequencing technology, makes possible relatively straight-forward chip-seq even on very scarce cell numbers e.g.
from stem cell populations.
this opens the door for extensive genome-wide investigation of the regulatory circuitries at all differentiation levels of in vivo tissues.

 genomic mapping of histone modifications, their writers, readers and erasers as well as transcription factors has become a house-hold approach to study the genome-wide regulation of gene expression programs. the most widely applied method to generate such global mapping data is chromatin immunoprecipitation coupled with high-throughput sequencing, which however generally requires millions of cells as input material. scarcity of cells in distinct, isolated in vivo populations such as phenotypically defined hematopoietic stem and progenitor cells has thus hampered direct experimentation on these. examining such sub-populations is of intense interest for the elucidation of mechanisms governing lineage choice and commitment as well as transcriptional de-regulation in disease. some researchers have cultured harvested cells to achieve sufficient cell numbers and performed chip-seq on cells undergoing differentiation in vitro, but this approach can give rise to biases due to culture conditions. recent advances in the methodology have demonstrated successful chip-seq on very limited cell numbers. however, these techniques are impeded by additional rounds of pre-amplification, potentially making them prone to introduce artifacts. one of these methods was established on cultured cells, while the other is limited to showing chip-seq with antibodies against histone modifications, yielding significantly more immunoprecipitated dna than chip with antibodies to transcription factors. furthermore, the complexity and cost of pre-amplification deters the implementation of the existing methods in many laboratories. another recent method uses an elegant step of pre-chip indexing of histone containing chromatin fragments, allowing downstream distinction of input material. the mixed inputs may thus function as mutual carriers during single-tube, small cell number chip. importantly, the indexing step selects against nucleosome-poor genomic regions, making this approach less useful for unbiased investigation of genome-wide tf occupancy. here, we describe a straightforward and versatile work-flow for both transcription factor and histone mark chip-seq on low abundance cell populations isolated directly from the in vivo setting. a key element is the introduction of bacterial carrier dna at the amplification step. this eliminates the previous need for pre-amplification and makes possible robust generation of sequencing libraries from picogram amounts of chip dna.  histone mark chip-seq of hematopoietic cell populations the scarcity of biologically relevant in vivo material is often barring global level investigations into normal development as well as the aberrant regulation behind cancer and other complex diseases. of particular interest are the genome-wide binding patterns of transcription factors and the associated epigenetic profiles, which may pinpoint aberrant molecular mechanisms underlying transcriptional dysregulation and development of disease. here, we use a standard facs regimen to isolate a specific hematopoietic gmp-blast population from cebpap30/p mice expressing a truncated variant of the myeloid transcription factor cebpa. these mice develop acute myeloid leukemia with complete penetrance, and have been studied in detail. however, the precise molecular dysregulation driving leukemogenesis remains obscure. we therefore developed a chip-seq assay compatible with the numbers of isolated leukemic cells from the in vivo context. first, we optimized our chip protocol for small cell numbers, which is described in detail here for clarity. immediately after the sorting procedure, isolated cells were exposed to formaldehyde for cross-linking chromatin-associated proteins to the dna, washed and snap frozen in liquid nitrogen. next, they were subjected to sonication to break the chromatin into suitably sized fragments. we found that careful inspection of the dna size distribution of each batch of chromatin was useful to prevent further processing of low quality samples. this was achieved either by processing a parallel sample of c-kit enriched bm cells, providing a sufficient cell number for standard gel electrophoresis, or by direct inspection of each sample using the bioanalyzer dna assay ). chromatin from roughly cells, equivalent to 250–300 ng of naked dna, was used as input for each chip experiment with antibodies against the histone marks h lys trimethylation or h lys trimethylation, performed in siliconized tubes with optimized washing conditions and titrated antibody and antibody-binding beads. utilizing a thorough approach of extended protein degradation and de-crosslinking steps, as well as phenol-chloroform extraction for retrieving chip dna ensured robust high recovery. this approach allowed us to effectively enrich for genomic sequences associated with either h3k27me or h3k4me as assessed by quantitative pcr. the h3k27me chip produced ca. 2 ng of dna for each sample. by making minor but important changes to the standard illumina protocol, we were able to consistently amplify the 2 ng chip dna to generate libraries for high-throughput sequencing. the h3k4me chip yielded an amount of dna below the effective range of standard absorbance or fluorescence assays. we circumvented this obstacle by taking advantage of the fluorescence nanodrop instrument, which allows reliable detection of dna down to 5 pg/ul in a ul sample volume. with this approach, h3k4me chip dna was measured to ca. 700 pg dna, which we pooled to obtain the 2 ng sufficient for robust amplification. using the illumina hiseq platform, we deep sequenced two libraries derived from two biologically independent samples for each of the two histone marks. we processed the aligned reads into genomic coverage profiles using standard procedures. visual analysis of the profiles suggested a good concordance with previous findings, showing enrichment of the h3k27me mark in gene bodies, intergenic regions as well as promoters and h3k4me in gene promoter regions. a quantitative analysis mapped h3k27me reads as 6% in promoter and 56% in gene body locations, while 21% of h3k4me reads resided in promoters. promoter h3k4me modifications were positively and h3k27me negatively correlated with activity of associated genes, as observed previously. finally, we assessed the reproducibility of our chip-seq approach by comparing coverage in promoter regions from two biologically independent replicates and found a high degree of correspondence, both by visual inspection and quantitative measures. in the examined cell type, we observe a near mutually exclusive pattern of h3k4me and h3k27me marks in promoters, as opposed to e.g. embryonic stem cells displaying a set of double-marked promoters at poised genes, a hall-mark of the undifferentiated state.figure generation of genomic coverage maps of dna-associated proteins from rare, isolated cell populations. schematic depiction of small-scale chip-seq workflow, including pico-gram dna quantification and addition of fragmented bacterial dna carrier. chip’ed dna in black, carrier-dna in light grey. validation of small-scale chip-seq histone mark maps. two h3k27me and h3k4me profiles and refseq gene positions across a 400 kb region on chromosome genomic distribution of all mapped reads, rounded numbers. scatterplots of promoter histone methylation and mrna expression in gmp-phenotype blast cells. each data point represents a single gene. quantile normalized. quantitative comparisons of promoter read coverage from two independent biological replicates of each histone modification using 1-kb tss centered bins. quantile normalized, 1/ diagonal indicated by dashed line. seqminer cluster heatmaps showing signal intensities for both replicates of h3k27me and h3k4me in a 20000 bp region centered on refseq tss positions. transcription factor chip-seq of specific, isolated cell populations chromatin-associated proteins with limited genomic distributions generally yield less chip dna when compared to chromatin components showing broader distributions. this is in line with our observations for h3k4me versus h3k27me modifications, with characteristic peak-like and broad distributions, respectively. accordingly, we expected transcription factor chip to yield even less output dna. to address this issue we used a larger input cell number. we performed chip with antibodies against cebpa, a transcription factor known to be expressed in the gmp blast population, using chromatin from or cells, corresponding to 500–1000 ng input dna per sample. by performing qpcr for amplicons covering known cebpa target sequences, we verified the quality of the immuno-precipitation step. utilizing the fluorescent nanodrop, we measured the chip dna yields of down to 250 pg, an amount prohibitive for standard use as a starting point for illumina amplification. we reasoned that an absolute loss during the amplification procedure could be ameliorated by adding a dna carrier. first, we set out to test the use of a synthetic 120-mer oligo devoid of the terminal 3’-oh group, which would prevent ligation to the amplification adaptors and hence preclude amplification during the pcr step. this gave rise to unacceptable biases as displayed by non-linear amplification of cebpa enriched regions, probably due to low complexity of the dna pool during pcr amplification. we next hypothesized that addition of complex carrier dna that could be amplified would be ideally suited to surpass this problem. hence, we performed an in silico mappability test of million 50-bp randomly extracted e. coli sequences, of which mapped to the mouse genome and to the human counterpart. adding fragmented bacterial dna to the cebpa chip dna produced the 2 ng established to perform robustly in amplification. by deep sequencing the resulting compound library, we were able to map up to 20% of the obtained sequences to the mouse genome. this corresponded with the ratio between chip dna and bacterial carrier and typical chip-seq mapping frequencies. cebpa peaks were observed in promoter proximal positions characteristic of many transcription factors. quantitative assessment of cebpa peak positions revealed a genomic distribution analogous to a thoroughly validated cebpa chip-seq dataset obtained with liver cells. further visual inspection identified several examples of cebpa peaks shared between hepatocyte and myeloid progenitor data sets. strikingly, the two archetypical homeostatic liver genes and carbamoyl-phosphate synthase ) displayed cebpa peaks in hepatocytes, but not in myeloid cells. conversely, many genes characteristic of the myeloid lineage displayed cebpa peaks only in the myeloid chip-seq dataset, colony stimulating factor receptor, matrix metallopeptidase, selectin l, colony stimulating factor, fc fragment of igg, low affinity iiib, receptor ). several of these are known cebpa targets. two additional pieces of evidence supported the validity and specificity of our cebpa genomic occupancy data. firstly, the top hit of a de novo motif search in the enriched sequences matched the known cebpa binding logo, and this motif was found strongly enriched in the peak centers. secondly, robust conservation in these sequences were centered on cebpa motifs, implying functional evolutionary constrain. visual and quantitative comparisons of coverage from two biologically independent repeats indicated a high degree of reproducibility. collectively, these data provide evidence that transcription factor chip-seq on small-cell-number populations is possible using bacterial dna as a carrier during the amplification step.figure validation of small-scale transcription factor chip-seq. profiles generated from pg cebpa chip-dna. region spanning a kb region on chromosome genomic distribution of top cebpa peaks from gmp-blasts compared with cebpa peak positions in hepatocytes, rounded numbers. cebpa coverage tracks from gmp-blast and hepatocyte gene loci of shared, specific liver or myeloid cell expression. see text for full gene names. de-novo motif search output from meme, and top match jaspar motif cebpa, both in logo format. data set as in d. distribution of the top match motif cebpa in bp regions centered on cebpa- peak summits. usf included for comparison. phylo-p conservation plots in bp and bp regions centered on cebpa-motifs in cebpa- peaks. green lines delineate motif position. quantitative comparison of mean read coverage of two chip-seq repeats, cebpa- and cebpa- using cebpa- peak regions defined by macs quantile normalized, 1/ diagonal indicated by dashed line. macs peak boundaries for cebpa- seqminer cluster heatmaps showing signal intensities for cebpa- and cebpa- profiles in bp regions centered on peak summits. for f and g, peak set as in b. amplification from picogram amounts of chip dna many immunophenotypically defined hematopoietic compartments, e.g. the hematopoietic stem cells, consist of a very limited number of cells. chip from small cell populations, as has been done previously by several groups, consistently yield very limited quantities of chip dna. hence, we wanted to investigate if our carrier dna amplification approach could be applied on pico-gram-scale amounts of dna. to test this, we generated a series of compound dna illumina amplified libraries with varying ratios of bacterial carrier dna and chip dna from three pooled cebpa chip samples to allow direct comparisons between libraries. aiming to minimize the dilution ratio, some of these libraries were generated using total input amounts of or 500 pg. the resulting four libraries, all displayed amplification output yields and size distributions comparable to libraries generated from 2 ng dna. high-throughput sequencing of these libraries resulted in mapping frequencies close to the expected based on standard chip-seq mapping efficiency and ratios of chip dna to bacterial carrier dna. e.g. from a read number of roughly million for cebpa- million mapped uniquely to the mouse genome. visually, genomic coverage profiles for each of the four new libraries closely matched our previous cebpa tracks. an analysis of the degree of correlation between dilute libraries and cebpa- indicated consistent, high reproducibility. strikingly, the library containing least chip dna display a pearson correlation of with the 300 pg cebpa- library and overall recapitulate the genomic coverage of this library.figure comparison of pico-scale transcription factor libraries. cebpa profiles of a kb region on chromosome based on progressively lower amounts of input chip-dna and bacterial carrier. quantitative comparisons of mean read coverage of cebpa- versus cebpa- /-4/-5/- chip-seq profiles in cebpa- peak regions defined by macs quantile normalized, 1/ diagonal indicated by dashed line. seqminer cluster heatmaps showing signal intensities for profiles cebpa- and cebpa- at bp regions centered on peak summits. for b and c, peak set as in figure 3b. next, we investigated the diluted libraries for presence of cebpa peaks at known cebpa targets, alpha and - beta, prostaglandin-endoperoxide synthase and interleukin receptor, alpha), and found these in all coverage profiles. to test our panel of libraries further, we performed qpcr to quantify amplicons corresponding to the cebpa enriched regions mentioned above, which indicated reproducible and practically uniform amplification across dilution ratios and input amounts.figure test of pico-scale chip-seq amplification linearity. cebpa profiles of promoter regions of six known cebp targets. qpcr validation of linearity. amplicons positioned in cebpa peak regions from a. chip-dna and bacterial carrier dna amounts indicated. chip-seq using cells to investigate if our methodology allows chip-seq on limited cell numbers, we transplanted a new cohort of mice with the leukemic strain also used in the above and facs-isolated batches of gmp-blasts. we optimized the chromatin preparation procedure to ensure maximal input material for the small-cell-number chip. thorough optimization of chip conditions produced robust enrichments for histone mark as well as tf chip, both of which compared favorably to chips done according to a previously published cell chip methodology by zwart et al.. the major advance in the previous study was addition of a combined mrna/histone carrier/blocker during the chip step. we amended our protocol to include such a carrier, resulting in even better enrichment as assessed by qpcr. a small amount of cell chip-dna was used for examining enrichment and establishing quantity, leaving ca. 60 pg dna. parallel generation of sequencing libraries with and without bacterial carrier demonstrated that at the 60 pg chip-dna range, carrier is required for robust production of high-quality illumina libraries. both visual inspection and quantitative analyses of genomic coverage tracks from cell chips revealed good correspondence with libraries generated from biologically independent higher-cell-number chips. cebpa- peaks were to a high degree shared with a biological repeat, the diluted library and the low-cell-number chip. importantly, the h3k4me3-10k profile generated from 60 pg chip-dna, which display substantially fewer mapped reads, is highly similar to the h3k4me3- profile from 2 ng dna resulting from chip without using bacterial carrier for amplification.figure cell histone mark and transcription factor chip-seq. chip using sorted gmp-blasts as input. our protocol with and without mrna/histone carrier, compared to the zwart protocol. qpcr assessment of enrichment ratios, n = 4- error bars show sd. *** = p < , t-test, holm-sidak correction for multiple testing. h3k4me3-chip, ratios are amplicon smc versus the negative sfi cebpa-chip, ratios are amplicon ptgs versus the negative sfi h3k4me and cebpa chip-seq profiles across a region spanning a 130 kb region on chromosome using or cells as input and cebpa chip-seq profiles of using or cells as input. quantitative comparisons of mean read coverage of h3k4me3- versus h3k4me3-10 k and cebpa- versus cebpa-10 k. all settings as described above. peak overlaps between alpha- and alpha- cebpa- and cebpa-10 k. macs peaks were ranked for fdr and the cebpa- top 1% set tested against peaks scoring fdr < 10− in the fdr top 1% or in top. schematic bar diagram depicting summed ngs-qc score for each of the listed small-scale chip-seq data sets. listed is number of cells used as input material, source of material and antibody target. the chip-dna fraction column denotes amplification of a fraction of dna from a higher-cell-number chip corresponding to the indicated number of cells. the carrier column indicates use of mrna/histone as carrier during the chip procedure. all scores represent best replicate. ngs-qc sum score is calculated as a sum of quartile scores for read sub-sampling of and 50% of total mapped reads. the recent establishment of the online next generation sequencing quality control generator, a useful cross-platform quality assessment tool, allowed a quantitative comparison of our chip-seq approach with already published methods. our methodology robustly produced ngs-qc scores matching or exceeding those from previous approaches. specifically, shankaranarayanan amplification resulted in considerably lower ngs-qc scores at the cell range, while adli amplification and the zwart methodology produced data sets with comparable scores for histone modification or transcription factor chip-seq profiles, respectively. discussion and  here, we present a complete work-flow for chip-seq on the limited cell populations of distinct in vivo cell compartments. by isolating a defined cell population with a standard immunophenotypic sorting regimen and using a rigorous and reliable chip approach, we were able to enrich regions marked by both broadly and more narrowly distributed histone modifications as well as the precise regions defined by occupancy of the central hematopoietic transcription factor, cebpa. importantly, the fluorescence nanodrop instrument allowed us to reliably measure the obtained picogram amounts of chip dna. we demonstrate that these amounts of chip dna could be faithfully amplified to generate libraries for illumina sequencing by addition of fragmented e. coli carrier dna. further, we show the resulting coverage profiles to match previous data for the actively transcribed gene associated and repressed gene associated histone marks, and to specifically recover known myeloid specific cebpa target regions. we provide evidence that cebpa bound regions from a pool of chip’ed dna down to 50 pg are amplified in a linear manner with our technique. finally, we demonstrate that our straight-forward methodology can produce high-quality chip-seq coverage tracks from as little as isolated in vivo cells using antibodies against histone mark as well as transcriptional factor antibodies. importantly, the h3k4me3-chip cell genomic coverage profile is very similar to the profiles generated without bacterial carrier, indicating that the addition of bacterial carrier does not impede amplification or illumina sequencing. our methodology can be used to elucidate important biological circuitries at a global level. for instance, we can clearly detect differences of direct targets of cebpa in specific cell types as illustrated in figure  importantly, two recently published studies demonstrate the usefulness of our approach by revealing molecular mechanisms behind initiation of acute myeloid leukemia and regulation of hematopoietic stem cells. furthermore, our technology should combine easily with other genomics approaches, for instance chia-pet, to permit generation of libraries from otherwise prohibitively small amounts of dna. other available methodologies that allow generation of chip-seq data from limited amounts of input material rely on an extra amplification step, based either on custom designed random primers or t rna-polymerase technology. even as these methods are very useful, introducing extra and complex steps in the amplification procedure will inevitably increase the risk of errors, and is costly and time-consuming. our method is based on the conceptually simple addition of non-mapping dna as carrier and should as such be easy to implement in any laboratory that already performs illumina platform chip-seq. a disadvantage of our approach is the added cost of sequencing e. coli dna, generating data that will be discarded. this drawback increases with the ratio between bacterial and chip dna, i.e. as fewer cells are used or less material recovered, for example as a result of poor antibodies or low expression of the chiped factor. we have tried to address this issue by demonstrating the feasibility of using just picogram total input material for the amplification procedure. the steady decline of sequencing prices should also help reduce this disadvantage. several groups have optimized chip from very small cell numbers, whereas our study focusses on refining the amplification step of chip-seq. some reports have shown the addition of carrier chromatin during the chip stage to facilitate the application on small cell numbers, but these methodologies are either not tested or incompatible with high-throughput sequencing. recently, zwart et al. demonstrated an increase in the chip-seq signal to noise ratio by adding carrier consisting of rna and histones. while chip protocols generally include bovine serum albumin as a carrier, zwart and coworkers speculate that combined oligonucleotide rna and histones mimic chromatin better, and hence offer improved blocking of spurious binding. both components are degraded prior to the amplification step, making the modification suitable for use in chip-seq. we compare a low-cell-number optimized version of our methodology and find it to surpass the zwart approach for the tested antibodies. nevertheless, adopting the mrna/histone chip level carrier considerably improves our protocol, clearly demonstrating the value of the zwart at al. contribution to the development of chip technology. in summary, our study, in combination with the progress of sequencing technology, makes possible relatively straight-forward chip-seq even on very scarce cell numbers e.g. from stem cell populations. this opens the door for extensive genome-wide investigation of the regulatory circuitries at all differentiation levels of in vivo tissues. 
chromatin-immunoprecipitation coupled with deep sequencing is used to map transcription factor occupancy and generate epigenetic profiles genome-wide. by employing non-mammalian genome mapping bacterial carrier dna during amplification, we reliably amplify down to 50 pg of chip dna from transcription factor and histone mark chip. chip-seqpico-scalehistone marktranscription factorepigenetic profileissue-copyright-statement© the author 2015 the requirement of nano-scale chip dna for generation of sequencing libraries has impeded chip-seq on in vivo tissues of low cell numbers. we further demonstrate that genomic profiles are highly resilient to changes in carrier dna to chip dna ratios. this represents a significant advance compared to existing technologies, which involve either complex steps of pre-selection for nucleosome-containing chromatin or pre-amplification of precipitated dna, making them prone to introduce experimental biases.
 chromatin-immunoprecipitation coupled with deep sequencing is used to map transcription factor occupancy and generate epigenetic profiles genome-wide. the requirement of nano-scale chip dna for generation of sequencing libraries has impeded chip-seq on in vivo tissues of low cell numbers.  we describe a robust, simple and scalable methodology for chip-seq of low-abundant cell populations, verified down to cells. by employing non-mammalian genome mapping bacterial carrier dna during amplification, we reliably amplify down to 50 pg of chip dna from transcription factor and histone mark chip. we further demonstrate that genomic profiles are highly resilient to changes in carrier dna to chip dna ratios.  this represents a significant advance compared to existing technologies, which involve either complex steps of pre-selection for nucleosome-containing chromatin or pre-amplification of precipitated dna, making them prone to introduce experimental biases. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords chip-seqpico-scalehistone marktranscription factorepigenetic profileissue-copyright-statement© the author 2015 