TITLE: low input ChIP-sequencing of  immune cells
AUTHORS: Wiebke Nahrendorf, Philip J Spence

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Cells can stably (and heritably) alter their gene expression profile through epigenetic modifications. Histones package DNA into chromatin and can be post-translationally modified - most prominently by methylation and acetylation. These histone modifications alter chromatin structure and DNA accessibility. We optimised a protocol for reliable high quality chromatin immunoprecipitation followed by DNA sequencing (ChIPseq) starting with just 60,000 monocytes isolated directly from mouse tissues by flow sorting. Our protocol can easily be adapted to other mouse or human cell types to interrogate the genome-wide distribution of histone modifications or transcription factor binding sites in immune cells directly </span><span style = "font-style:italic;">ex vivo</span><span style = "font-style:italic;">.</span></div></div>

[STEPS]
?. [DNA-Protein crosslinking and cell sorting]
prepare single cell suspensions and lyse erythrocytes at  RECOMMENDATION: work in 15 ml sterile, RNAse/ DNAse free, non pyrogenic polypropylene conical tubes until step 11.NOTE: this protocol was optimised to isolate monocytes from mouse spleens and bone marrow - but it can be easily adapted to  work with most other mouse and human tissues and cell types. adhere to best practice for your tissue when preparing single cells suspensions and lysing erythrocytes.
4 Room temperature
?. [DNA-Protein crosslinking and cell sorting]
count cells, then Fc block and antibody stain in FLOW BUFFER* (scale appropriately: stain max 2x107 cells in ) for  at * Buffers and Solutions in bold capitals are described in detail in Materials sectionNOTE: design, titrate and test your antibody panel carefully beforehand.
1 mL
4 Room temperature
?. [DNA-Protein crosslinking and cell sorting]
wash cells twice in PBS
Centrifuge: 350 34, 5 min
?. [DNA-Protein crosslinking and cell sorting]
gently resuspend  cells in FIXATION BUFFER* ( for every 1x107 cells) CRITICAL: warm FIXATION BUFFER to  before use.
1 mL
0 Room temperature
?. [DNA-Protein crosslinking and cell sorting]
incubate for exactly  at , gently flick to mix occasionally
0 Room temperature
?. [DNA-Protein crosslinking and cell sorting]
add  QUENCH BUFFER  (final Glycine concentration : for every  FIXATION BUFFER added in step 4 add  QUENCH BUFFER)
4 Room temperature
1 mL
50 µl
?. [DNA-Protein crosslinking and cell sorting]
incubate for  at ,  gently flick to mix occasionally
0 Room temperature
?. [DNA-Protein crosslinking and cell sorting]
NOTE: faster, longer centrifugations going forward, since cell velocity changes after fixation.
Centrifuge: 450 34, 10 min, 4 10
?. [DNA-Protein crosslinking and cell sorting]
aspirate supernatant carefully (leave approx.  ) and resuspend cells in  cold PBS
100 µl
12 mL
?. [DNA-Protein crosslinking and cell sorting]
Centrifuge: 450 34, 10 min, 4 10
?. [DNA-Protein crosslinking and cell sorting]
aspirate supernatant carefully (leave approx.  ) and resuspend cells in  cold FLOW BUFFER
100 µl
3 mL
?. [DNA-Protein crosslinking and cell sorting]
SORT 60,000 desired cells on BD FACS Aria III or similar cell sorter (85 um nozzle, sort precision mode: purity, sample and collection chamber  ) into 5 ml polypropylene FACS tubes with  COLLECTION BUFFER.RECOMMENDATION: sort several technical replicates from one biological sample and chromatin-immunoprecipitate each replicate with an antibody against a different histone modifications. in this way you will get a more detailed picture of the epigenetic landscape within each biological sample.NOTE: always perform a test sort beforehand, where you set up all parameters, gates and compensation ready for your big ChIPseq sort day. check cell recovery - some cell types are very fragile and may require alteration of sort or collection parameters for optimal viability. our recovery was 40 - 60 % of sorted cells i.e. we continue the protocol with approx 30,000 cells. always check the purity of your sort before and after your last sample (and in between if you encountered any problems): > 95% of sorted cells should fall in the gates for your population of interest and debris should be minimal.
4 °C
2.5 mL
?. [DNA-Protein crosslinking and cell sorting]
Centrifuge: 450 34, 10 min, 4 10
?. [DNA-Protein crosslinking and cell sorting]
resuspend in  cold HBSS + protease inhibitors (PIC) +  sodium butyrate
2 mL
?. [DNA-Protein crosslinking and cell sorting]
Centrifuge: 450 34, 10 min, 4 10
?. [DNA-Protein crosslinking and cell sorting]
flash freeze cell pellet in methanol bath on dry ice and store at  for up to 3 months
-80 °C
?. [DNA-Protein crosslinking and cell sorting]
aspirate supernatant carefully (leave approx  behind)
50 µl
?. [DNA-Protein crosslinking and cell sorting]
aspirate supernatant carefully (leave approx  behind)
50 µl
?. [cell lysis and chromatin shearing]
the following section uses reagents from the True MicroChIP kit (Diagenode, #C01010130) with a modified protocolNOTE: work in area designated for low input DNA work, use designated pipettes with sterile RNAse and DNAse free tips, clean area and pipettes as well as all other equipment with 1 % Distel, 70 % Ethanol and DNAZap before starting.
?. [cell lysis and chromatin shearing]
thaw samples slowly  and add  ice-cold HBSS + PIC
on ice
1 mL
?. [cell lysis and chromatin shearing]
Centrifuge: 450 34, 10 min, 4 10
?. [cell lysis and chromatin shearing]
equilibrate lysis buffer tL1 to  (all crystals should be dissolved) and add protease inhibitor cocktail (PIC, from True MicroChIP Kit) for 1 sample:   tL1 +  PIC (1:200)
0 Room temperature
25 µl
0.125 µl
?. [cell lysis and chromatin shearing]
add  tL1 with PIC to the cell pellet – gently vortex to resuspend and flick until bubbles form
25 µl
?. [cell lysis and chromatin shearing]
incubate for
on ice
?. [cell lysis and chromatin shearing]
add  HBSS with PIC, mix by pipetting and transfer to 1.5 ml Bioruptor Pico microtubes
75 µl
?. [cell lysis and chromatin shearing]
using the Bioruptor Pico sonicate for 5 cycles 30 sec ON 30 sec OFF to shear the chromatin.NOTE: sonication time and intervals are unique for each cell type. for optimal ChIPseq chromatin should be sheared into 100 - 300 bp fragments. see [QC chromatin shearing] in step 30.1 for how to optimise shearing.
?. [cell lysis and chromatin shearing]
briefly vortex and place
on ice
?. [cell lysis and chromatin shearing]
Centrifuge: 14000 34, 10 min, 4 10
?. [cell lysis and chromatin shearing]
transfer supernatant (= sheared chromatin) to 1.5 ml DNA LoBind tubesheared chromatin can be stored at  for up to 8 weeks or immediately immunoprecipitated (see next section)
100 µl
-80 °C
?. [cell lysis and chromatin shearing]
aspirate supernatant carefully (leave as little behind as possible), keep pellets
on ice
?. [cell lysis and chromatin shearing]
OPTIONAL: optimise Chromatin shearing for cell type of interest [uses one flow-sorted technical replicate]use reagents from the True MicroChIP kit (Diagenode, #C01010130) with a modified protocol. during optimisation it may pay off to use the designated Chromatin shearing optimization kit – high SDS (Diagenode, #C01020012)
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							start with  sheared chromatin in 1.5 ml DNA LoBind tubedilute RNase cocktail ( +  DEPC-treated water) and add  to the sheared chromatinincubate  at add  elution buffer tE1 and  elution buffer tE2, mix thoroughly by pipettingdecrosslink proteins from DNA for at least  or   in a ThermoMixer (1300 rpm) at spin tubes brieflyadd      Phenol/Chloroform/Isoamyl alcohol 25:24:1vortex for  , incubate for   on rotating wheel at   transfer aqueous phase to new 1.5 ml DNA LoBind tubeadd     Chloroform/Isoamyl alcohol 24:1vortex for  , incubate for   on rotating wheel at    transfer aqueous phase to new 1.5 ml DNA LoBind tube ( approx  )to precipitate the DNA add:    add   Phenol/Chloroform/Isoamyl alcohol 25:24:1vortex for , incubate for  on rotating wheel at transfer aqueous phase to new 1.5 ml DNA LoBind tubeadd     Chloroform/Isoamyl alcohol 24:1vortex for  , incubate for   on rotating wheel at    transfer aqueous phase to new 1.5 ml DNA LoBind tube ( approx  )to precipitate the DNA add:    add  Chloroform/Isoamyl alcohol 24:1vortex for , incubate for  on rotating wheel at transfer aqueous phase to new 1.5 ml DNA LoBind tube ( approx ) tP1 tCP1 tCP2 ice cold 100 % Ethanol16. incubate at   for 17. 18. carefully discard supernatant and add  ice cold 70 % Ethanol19.  20. carefully remove all supernatant and allow pellet to air-dry for max. 21. resuspend pellet in  TE: to assess DNA fragment size distribution and integrity use  for  Bioanalyzer HS DNA Chip (see step 113 for details) and  to run on a 1.5 % Agarose gel at 100 V for  (use a 100 kb ladder)22. stain and assess gel image
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100 µl
1 µl
150 µl
2 µl
37 °C
100 µl
8 µl
65 °C
200 µl
65 Room temperature
65 Room temperature
Centrifuge: 14000 34, 2 min
200 µl
0 Room temperature
65 Room temperature
Centrifuge: 14000 34, 2 min
150 µl
15 µl
2 µl
2 µl
1 µl
-80 °C
Centrifuge: 14000 34, 25 min, 4 10
500 µl
Centrifuge: 14000 34, 10 min, 4 10
12 µl
1 µl
10 µl
sheared chromatin fragments should be between 100 and 300 bp
?. [chromatin immunoprecipitation (ChIP)]
the following section uses reagents from the True MicroChIP kit with a modified protocolNOTE: work in area designated for low input DNA work, use designated pipettes with sterile RNAse and DNAse free tips, clean area and pipettes as well as all other equipment with 1 % Distel, 70 % Ethanol and DNAZap before starting.
?. [chromatin immunoprecipitation (ChIP)]
add  tC1 + PIC to  sheared chromatin
100 µl
100 µl
?. [chromatin immunoprecipitation (ChIP)]
OPTIONAL: to normalise for technical variation between samples from this point onwards spike in a small amount of Drosophila melanogaster chromatin into each sample. an antibody against the Drosophila-specific histone variant H2Av then reliably pulls down a fraction of the Drosophila chromatin. this should happen consistently across all samples. after sequencing, the ratio of data mapping to the Drosophila genome vs your organisms genome creates a normalisation factor for each sample. you can then normalise your experimental tag counts by this factor. for more information: https://www.activemotif.com/catalog/1091/chip-normalizationadd  Drosophila spike-in chromatin(if you do this also add  spike-in antibody in step 36)
140 pg
0.3 µg
?. [chromatin immunoprecipitation (ChIP)]
CRITICAL: remove  (5 %) of sheared chromatin as input sample, store at  in a 1.5 ml DNA LoBind tube. NOTE: input samples (one for each biological replicate) are essential to analyse ChIPseq. it is the measurement of epigenetic landscape in your cells before immunoprecipitation - all enrichment is measured relative to it.
10 µl
4 °C
?. [chromatin immunoprecipitation (ChIP)]
add your antibody of interest to the remaining 95% of sheared chromatin for immunoprecipitation: we used antibodies against H3K27ac (), H3K4me1 () and H3K9me3 () to investigate activation and repression of transcription as well as the future potential to respond to stimuli.H3K27ac marks transcription start sites to activate transcriptionH3K4me1 marks enhancers and superenhancers to promote gene expression H3K9me3 condenses DNA into heterochromatin to silence gene expressionNOTE: other ChIPseq-grade antibodies (for example against transcription factors) can be used. titrate all antibodies for optimal ChIPseq. some protocol recommend qPCR for validating titrations; however, we find that qPCR results do not predict ChIP sequencing outcome. we instead recommend a test sequencing run to validate antibodies and the concentrations they are used at.OPTIONAL: add  spike-in antibody (see step 34)
2 µg
5 µg
1 µg
0.3 µg
?. [chromatin immunoprecipitation (ChIP)]
incubate  on a rotating wheel (40 rpm) in the cold room
4 °C
?. [chromatin immunoprecipitation (ChIP)]
next morning: prepare DiaMag Protein A-coated magnetic beadsfor 1 sample mix  beads (pipette up and down > 20 times to get an even suspension) with  beads wash buffer tBW1 in a 1.5 ml tube
10 µl
50 µl
?. [chromatin immunoprecipitation (ChIP)]
place in the DynaMag- 2 magnet and wait for
?. [chromatin immunoprecipitation (ChIP)]
discard the supernatant (keep tube in magnet)
?. [chromatin immunoprecipitation (ChIP)]
take tube out of magnet and gently resuspend the beads in  tBW1
50 µl
?. [chromatin immunoprecipitation (ChIP)]
place in the magnet and wait for
?. [chromatin immunoprecipitation (ChIP)]
discard the supernatant (keep tube in magnet)
?. [chromatin immunoprecipitation (ChIP)]
take tube out of magnet and gently resuspend the beads in  tBW1
10 µl
?. [chromatin immunoprecipitation (ChIP)]
remove samples from rotating wheel (keep ) and spin briefly to collect all liquid in the bottom of the tube
on ice
?. [chromatin immunoprecipitation (ChIP)]
add  of washed beads
10 µl
?. [chromatin immunoprecipitation (ChIP)]
incubate for  on a rotating wheel (40 rpm) in the cold room
4 °C
?. [washes]
the following section uses reagents from the True MicroChIP kit with a modified protocolthorough, careful washing is key for high quality ChIPseq, since it removes non-antibody bound chromatin fragments and therefore reduces background.NOTE: work in area designated for low input DNA work, use designated pipettes with sterile RNAse and DNAse free tips, clean area and pipettes as well as all other equipment with 1 % Distel, 70 % Ethanol and DNAZap before starting.
?. [washes]
place your samples in the magnet and wait for  - the beads (and the immunoprecipitated chromatin bound to them) will bind to the side of the tube facing the magnet
?. [washes]
discard the supernatant (keep tube in magnet)
?. [washes]
take tube out of magnet and gently resuspend the beads in  ice cold wash buffer tW1NOTE: do not create bubbles.
100 µl
?. [washes]
incubate for  on a rotating wheel (40 rpm) in the cold room
4 °C
?. [washes]
remove samples from rotating wheel (keep ) and spin briefly to collect all liquid in the bottom of the tube
on ice
?. [DNA decrosslinking]
the following section uses reagents from the True MicroChIP kit with a modified protocolNOTE: work in area designated for low input DNA work, use designated pipettes with sterile RNAse and DNAse free tips, clean area and pipettes as well as all other equipment with 1 % Distel, 70 % Ethanol and DNAZap before starting.
?. [DNA decrosslinking]
after removing wash buffer tW4 take tube out of magnet and gently resuspend the beads in  elution buffer tE1 (equilibrate to  before use - tE1 should be a clear solution)
200 µl
4 Room temperature
?. [DNA decrosslinking]
take the input samples (10 μl) you saved in step 35 out of the fridge and add  elution buffer tE1
190 µl
?. [DNA decrosslinking]
incubate both ChIP and input samples for  on a rotating wheel (40 rpm) at
37 Room temperature
?. [DNA decrosslinking]
remove samples from rotating wheel and spin briefly to collect all liquid in the bottom of the tube
?. [DNA decrosslinking]
place ChIP samples in the  magnet and wait for
-20 Room temperature
?. [DNA decrosslinking]
ChIP samples: keep the tube in the magnet and transfer the supernatant (= your immunoprecipitated chromatin) to a new 1.5 ml DNA LoBind tube
?. [DNA decrosslinking]
add  elution buffer tE2 to both ChIP and input samples
8 µl
?. [DNA decrosslinking]
decrosslink proteins from DNA (for both ChIP and input samples)  in a ThermoMixer (1300 rpm) at
65 °C
?. [DNA purification using Micro ChIP DiaPure columns]
the following section uses Micro ChIP DiaPure columns (Diagenode, #C03040001) according to manufacturers instructionsNOTE: work in area designated for low input DNA work, use designated pipettes with sterile RNAse and DNAse free tips, clean area and pipettes as well as all other equipment with 1 % Distel, 70 % Ethanol and DNAZap before starting.
?. [DNA purification using Micro ChIP DiaPure columns]
add  (5 Vol)  ChIP DNA binding buffer and mix gently by pipetting
1000 µl
0 Room temperature
?. [DNA purification using Micro ChIP DiaPure columns]
transfer  to the spin column in its collection tube
600 µl
?. [DNA purification using Micro ChIP DiaPure columns]
Centrifuge: 10000 34, 30 s
?. [DNA purification using Micro ChIP DiaPure columns]
discard the flow-through
?. [DNA purification using Micro ChIP DiaPure columns]
transfer the remaining  to the spin column in its collection tube
600 µl
?. [DNA purification using Micro ChIP DiaPure columns]
Centrifuge: 10000 34, 30 s
?. [DNA purification using Micro ChIP DiaPure columns]
discard the flow-through
?. [DNA purification using Micro ChIP DiaPure columns]
add  DNA wash bufferCRITICAL: make sure Ethanol was added to the buffer.
200 µl
0 Room temperature
?. [DNA purification using Micro ChIP DiaPure columns]
Centrifuge: 10000 34, 30 s
?. [DNA purification using Micro ChIP DiaPure columns]
add  DNA wash buffer
200 µl
0 Room temperature
?. [DNA purification using Micro ChIP DiaPure columns]
Centrifuge: 10000 34, 30 s
?. [DNA purification using Micro ChIP DiaPure columns]
transfer the column to a new 1.5 ml DNA LoBind tube
?. [DNA purification using Micro ChIP DiaPure columns]
to elute the DNA add  DNA elution buffer directly onto the column matrix and incubate for  at
15.2 µl
0 Room temperature
65 Room temperature
?. [DNA purification using Micro ChIP DiaPure columns]
then discard the column and transfer tube with DNA
Centrifuge: 10000 34, 30 s
on ice
?. [DNA purification using Micro ChIP DiaPure columns]
DNA can be stored at  before library preparation for up to 2 weeks
-20 °C
?. [Library preparation]
the following section uses the Diagenode MicroPlex Library preparation kit v2  (Diagenode, #C05010012) according to manufacturers instructionsNOTE: work in area designated for library preperation (distinct from low input DNA area), use designated library preparation pipettes with sterile RNAse and DNAse free tips, clean area and pipettes as well as all other equipment with 1 % Distel, 70 % Ethanol and DNA-ZAP before starting.NOTE:  keep samples and reagents  throughout.
on ice
?. [Library preparation]
in clear 96 well Lightcycler 480 plate mix  ChIP-ed, purified DNA with  template preparation buffer and  template preparation enzyme
10 µl
2 µl
1 µl
?. [Library preparation]
gently mix by pipetting, cap using strip lids and spin briefly to collect all liquid in the bottom of the wells
?. [Library preparation]
run on a standard PCR machine (settings: plate, 13 μl Volume, heated lid)  AB1temperaturetime222 °C25 min355 °C20 min44 °C∞
AB1temperaturetime222 °C25 min355 °C20 min44 °C∞
?. [Library preparation]
transfer plate back  as soon as PCR machine has cooled to , spin briefly to collect all liquid in the bottom of the wells
on ice
4 °C
?. [Library preparation]
carefully open lids and add  library synthesis buffer and library synthesis enzyme
1 µl
1 µl
?. [Library preparation]
gently mix by pipetting, cap using strip-lids and spin briefly to collect all liquid in the bottom of the tube
?. [Library preparation]
incubate once more using the same standard PCR machine (settings: plate, 15 μl Volume, heated lid)  AB1temperaturetime222 °C40 min34 °C∞
AB1temperaturetime222 °C40 min34 °C∞
?. [Library preparation]
transfer plate back  as soon as samples PCR machine has cooled to, spin briefly to collect all liquid in the bottom of the wells
on ice
4 °C
?. [Library preparation]
carefully open lids and add  library amplification master mix:  AB1reagentvolume/reaction2Library amplification buffer25 μl3Library amplification enzyme1 μl4EvaGreen2.5 μl5Nuclease free water1.5 μl
30 µl
AB1reagentvolume/reaction2Library amplification buffer25 μl3Library amplification enzyme1 μl4EvaGreen2.5 μl5Nuclease free water1.5 μl
?. [Library preparation]
add  indexing reagent (total volume 50 μl) - to avoid cross-contamination spray index lid with DNAZap and wipe dry, change gloves after each indexNOTE: carefully consider your sequencing requirements and plan which/how many libraries you are going to pool in each lane and index samples accordingly. see below for details of the standard Illumina indices supplied with the MicroPlex Library preparation kit v2 (12 indices, Diagenode #C05010012,). a kit with 48 indices is also available: MicroPlex Library Preparation Kit v2 (48 indexes, Diagenode #C05010014).  ABC1index numberindex IDindex sequence21iPCRtagT1ATCACGTT32iPCRtagT2CGATGTTT43iPCRtagT3TTAGGCAT54iPCRtagT4TGACCACT65iPCRtagT5ACAGTGGT76iPCRtagT6GCCAATGT87iPCRtagT7CAGATCTG98iPCRtagT8ACTTGATG109iPCRtagT9GATCAGCG1110iPCRtagT10TAGCTTGT1211iPCRtagT11GGCTACAG1312iPCRtagT12CTTGTACT
5 µl
ABC1index numberindex IDindex sequence21iPCRtagT1ATCACGTT32iPCRtagT2CGATGTTT43iPCRtagT3TTAGGCAT54iPCRtagT4TGACCACT65iPCRtagT5ACAGTGGT76iPCRtagT6GCCAATGT87iPCRtagT7CAGATCTG98iPCRtagT8ACTTGATG109iPCRtagT9GATCAGCG1110iPCRtagT10TAGCTTGT1211iPCRtagT11GGCTACAG1312iPCRtagT12CTTGTACT
?. [Library preparation]
gently mix by pipetting, use sealing foil and
Centrifuge: 1300 34, 2 min, 4 10
?. [Library preparation]
run on real time quantitative PCR machine (Roche Lightcycler 480) to monitor library amplification  ABCD1temperaturetimeramp rate2extension72 °C3 min3° C/sec3cleavage85 °C2 min3° C/sec4denaturation95 °C2 min3° C/sec56addition of indices98 °C20 sec3° C/sec767 °C20 sec2.2 °C/sec872 °C40 sec 3 °C/sec9repeat steps 6 to 8 four times1011library amplification98 °C20 sec3 °C/sec1272 °C50 sec: record fluorescence using "single acquisition"2.2 °C/sec13repeat steps 11 & 12 for x* number of cycles1415cool - hold37 °C **1 h2.2 °C/sec
ABCD1temperaturetimeramp rate2extension72 °C3 min3° C/sec3cleavage85 °C2 min3° C/sec4denaturation95 °C2 min3° C/sec56addition of indices98 °C20 sec3° C/sec767 °C20 sec2.2 °C/sec872 °C40 sec 3 °C/sec9repeat steps 6 to 8 four times1011library amplification98 °C20 sec3 °C/sec1272 °C50 sec: record fluorescence using "single acquisition"2.2 °C/sec13repeat steps 11 & 12 for x* number of cycles1415cool - hold37 °C **1 h2.2 °C/sec
?. [chromatin immunoprecipitation (ChIP)]
add protease inhibitor cocktail (True MicroChIP Kit) to Chip buffer tC1 for 1 sample:  tC1 +  PIC
100 µl
0.5 µl
?. [washes]
remove samples from rotating wheel (keep ) and spin briefly to collect all liquid in the bottom of the tube
on ice
?. [washes]
place magnet   and keep samples and all buffers ice-cold throughout
on ice
?. [DNA purification using Micro ChIP DiaPure columns]
spin decrosslinked samples (ChIP and input samples: 200 μl Volume) briefly to collect all liquid in the bottom of the tube
?. [Library preparation]
use  of amplified library to quantify the amount of DNA with Qubit dsDNA HS assay Kit according to manufacturers instruction
1 µl
5 - 15 ng/ μl if concentration is significantly lower return sample to real time PCR machine for extra amplification
?. [Library preparation]
transfer library from plate to 1.5 ml DNA LoBind tube (keep )NOTE: libraries (individual or pooled) can be stored at  for up to 2 weeks
on ice
-20 °C
?. [Library preparation]
dilute  of amplified library in  TE to asses DNA intergrity and size distribution using Bioanalyzer High Sensitivity DNA Kit according to manufacturers instructions
1 µl
4 µl
bell curve 200 - 2000 bp, average size approx. 400 bp
?. [Library preparation]
pool libraries with different indices at equal molarities in 1.5 ml DNA LoBind tube (aim to reach a volume just over )
100 µl
molecular mass of dsDNA = 660 [g/mol/bp]calculation:concentration [ng/ul] * 106 * 1/660 * 1/average size [bp] = molarity [nM]
?. [Library purification using AMPure beads]
NOTE: work in area designated for library preperation (distinct from low input DNA area), use designated library preparation pipettes with sterile RNAse and DNAse free tips, clean area and pipettes as well as all other equipment with 1 % Distel, 70 % Ethanol and DNA-ZAP before starting.
?. [Library purification using AMPure beads]
bring  pooled libraries, AMPure beads and freshly prepared 80 % Ethanol to
-20 Room temperature
?. [Library purification using AMPure beads]
resuspend AMPure beads until homogenous solution and add  to of pooled library (1:1 ratio); mix until homogenous
100 µl
100 µl
?. [Library purification using AMPure beads]
incubate at  for
-20 Room temperature
?. [Library purification using AMPure beads]
spin briefly () to collect all liquid in the bottom of the tube
?. [Library purification using AMPure beads]
place tube in DynaMag- 2 magnet, wait for  until all beads are bound (solution clear)
?. [Library purification using AMPure beads]
discard supernatant
?. [Library purification using AMPure beads]
add  80 % Ethanol
300 µl
?. [Library purification using AMPure beads]
rotate the tube clockwise by 90 °, wait for  and repeat 3 more times
?. [Library purification using AMPure beads]
discard supernatant
?. [Library purification using AMPure beads]
add  80 % Ethanol
300 µl
?. [Library purification using AMPure beads]
rotate the tube clockwise by 90 °, wait for  and repeat 3 more times
?. [Library purification using AMPure beads]
discard supernatant
?. [Library purification using AMPure beads]
spin briefly () to collect all liquid in the bottom of the tube
?. [Library purification using AMPure beads]
place tube in DynaMag- 2 magnet, wait for
?. [Library purification using AMPure beads]
remove all residual Ethanol
?. [Library purification using AMPure beads]
remove tube from magnet and dry the beads with lid open for max  in ThermoMixer ()
37 °C
?. [Library purification using AMPure beads]
resuspend the beads in  TE, spin briefly () to collect all liquid in the bottom of the tube
50 µl
?. [Library purification using AMPure beads]
place tube in magnet, wait for
?. [Library purification using AMPure beads]
carefully transfer the eluted DNA to a new 1.5 ml DNA LoBind tube
?. [Library purification using AMPure beads]
use  of pooled purified library to quantify the amount of DNA with Qubit dsDNA HS assay Kit according to manufacturers instruction (see step 112 for details)
1 µl
expect 10 ng/ μl (i.e. a total of 500 ng in 50 μl)
?. [Library purification using AMPure beads]
dilute  of pooled purified library in  TE to asses integrity and size distribution of libraries using Bioanalyzer High Sensitivity DNA Kit according to manufacturers instructions (see step 113 for details)
1 µl
4 µl
bell curve 200 - 2000 bp (all small fragments removed by AMPure XP bead purification), average size approx. 400 bp
?. [Library purification using AMPure beads]
pooled, purified libraries can be stored at  before sequencing for up to 2 months
-20 °C
our samples were sequenced by Edinburgh Genomics https://genomics.ed.ac.uk  on the Illumina NovaSeq S1 yielding approx 750 x 106 100 bp paired end reads per lane. we aimed for a depth of 70 x 106 paired end reads for each sample. ChIPed samples (for all different histone modifications) and matched input sample should be sequenced on the same lane. we used the motif discovery software HOMER for data anlaysis (http://homer.ucsd.edu/homer/). our ChIPseq data is publicly available: GEO accession number GSE150478.
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							NOTE: we subscribe to the notion that ChIPseq is qualitative (it can reveal the presence or absence of a histone modification at a particular genomic location) - not quantitative (it does not reveal biologically meaningful differences in peak height, which are often influenced by the efficiency of immunoprecipitation). please consider this when analysing your results.
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?. [washes]
remove samples from rotating wheel (keep ) and spin briefly to collect all liquid in the bottom of the tube
on ice
?. [washes]
place your samples in the magnet and wait for
?. [washes]
discard the supernatant (keep tube in magnet)
?. [washes]
take tube out of magnet and gently resuspend the beads in  ice cold wash buffer tW2NOTE: do not create bubbles.
100 µl
?. [washes]
incubate for  on a rotating wheel (40 rpm) in the cold room
4 °C
?. [washes]
remove samples from rotating wheel (keep ) and spin briefly to collect all liquid in the bottom of the tube
on ice
?. [washes]
place your samples in the magnet and wait for
?. [washes]
discard the supernatant (keep tube in magnet)
?. [washes]
take tube out of magnet and gently resuspend the beads in  ice cold wash buffer tW3NOTE: do not create bubbles.
100 µl
?. [washes]
incubate for  on a rotating wheel (40 rpm) in the cold room
4 °C
?. [washes]
remove samples from rotating wheel (keep ) and spin briefly to collect all liquid in the bottom of the tube
on ice
?. [washes]
place your samples in the magnet and wait for
?. [washes]
discard the supernatant (keep tube in magnet)
?. [washes]
take tube out of magnet and gently resuspend the beads in  ice cold wash buffer tW4NOTE: do not create bubbles.
100 µl
?. [washes]
incubate for  on a rotating wheel (40 rpm) in the cold room
4 °C
?. [washes]
place your samples in the magnet and wait for
?. [washes]
discard the supernatant (keep tube in magnet)