TITLE: MINUTE ChIP
AUTHORS: Banushree Kumar, Simon Elsässer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">The need for quantitative ChIP-seq has been widely appreciated, and a number of quantitative methods have been proposed. Mint-ChIP, developed by the Bernstein lab provides a streamlined one-pot chromatin barcoding and a post ChIP linear amplification that requires only one adaptor per chromatin fragment. Here, we introduce unique molecule (unique molecular identifier [UMI]) counting and paired-end mapping of the chromatin fragments to this method, which we then termed MINUTEChIP for multiplexed indexed unique molecule T7 amplification end-to-end sequencing. For paired-end mapping, we modified the linear amplification strategy introduced in Mint-ChIP by priming the cDNA synthesis from a 30 adaptor (RA3) ligated onto the amplified RNA, thus maintaining the original genomic fragment length, retrieving atypical mononucleosomal fragment length for histone ChIP. We have also optimized the ligation conditions and stoichiometry of adaptors to chromatin to enable sequencing of the input. We show that MINUTE-ChIP is a sensitive method with a large linear dynamic range for accurately quantifying relative differences in genome-wide histone modification patterns across multiple pooled samples.</div></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/ab67304d0fec75ebfd89851646ff1c06fbc3769cef7bdba0163c5951dcb9b10d/btfa9xxw.png" /></div></div>

[STEPS]
?. [LYSIS AND DIGESTION]
Harvest 10^6 cells, spin, flash freeze the pellet.
Make sure cell pellet is frozen with as little excess liquid as possible
?. [LYSIS AND DIGESTION]
On the day of experiment resuspend in  of ice cold PBS.
50 µl
?. [LYSIS AND DIGESTION]
Add of MNase mix (final 2U/ul in 2xLyB for mouse embryonic stem cells).
50 µl
MNase mix :First add 20 μl protease inhibitor to 1 ml 2x lysis buffer (2xLyB)Stepwise dilute MNase (M0247S NEB 2000 U/ul ) to 20U/μl and then 2U/μl in 2x LyBFinal working conc. = 2U/μl in 2xLyB.
?. [LYSIS AND DIGESTION]
Lyse cells on ice for . Start preparing the enzyme mix for T7 adapter ligation.
?. [LYSIS AND DIGESTION]
Digest at  for  with agitation at 800 rpm.
37 °C
?. [LYSIS AND DIGESTION]
Transfer tubes
on ice
?. [T7 ADAPTER LIGATION]
Prepare enzyme mix:  AB1Per 80 μl rxn230 mM EGTA (0.5 M)4.831× buffer (10×)8.041.5 mM ATP (10 mM)12.051× dNTP mix (10×)8.06End-Repair Enzyme mix1.67Fast-Link DNA ligase1.685 mM sodium butyrate (1 M)0.291× Protease inhibitor (100×)0.410Total volume36.6 μl
AB1Per 80 μl rxn230 mM EGTA (0.5 M)4.831× buffer (10×)8.041.5 mM ATP (10 mM)12.051× dNTP mix (10×)8.06End-Repair Enzyme mix1.67Fast-Link DNA ligase1.685 mM sodium butyrate (1 M)0.291× Protease inhibitor (100×)0.410Total volume36.6 μl
?. [T7 ADAPTER LIGATION]
QUICKLY add enzyme mix to  of the digested cells. Then add  of unique T7 adapter to each sample.Total volume ~80 μl (40 μl cells + 4 μl T7 adapter + 36 μl enzyme mix)
36.6 µl
40 µl
4 µl
?. [T7 ADAPTER LIGATION]
Next day, add 1x protease inhibitor (100x) to Lysis dilution buffer and add of it to each sample in order to end reaction (Total volume per well: )
80 µl
160 µl
?. [IMMUNOPRECIPITATION]
Precouple antibody to the magnetic beads
IP conditions such as amount of antibody to use, volume of pooled lysate to be addedand duration of incubation can be optimized for individual experiments.
?. [IMMUNOPRECIPITATION]
Was  Protein G/A beads  with 2x  PBS-T(0.1%)
50 µl
1000 µl
?. [IMMUNOPRECIPITATION]
Resuspend beads in  of the same buffer and incubate with appropriate amountof antibodies for  at
200 µl
0 Room temperature
?. [IMMUNOPRECIPITATION]
Wash coupled beads quickly ( RIPA and resuspend in  of Lysisdilution buffer.
200 µl
50 µl
?. [IMMUNOPRECIPITATION]
Top up with 200 - 400 μl of the pooled lysate for each IP*, and rotate tube for  at
4 °C
?. [IMMUNOPRECIPITATION]
Wash
?. [IMMUNOPRECIPITATION]
Magnetize samples and save supernatant at -20ºC
This supernatant can be used for checking IP efficiency or other troubleshooting
?. [IMMUNOPRECIPITATION]
Resuspend, rotate for 5 mins, magnetize and aspirate :2x with 500 μl ice cold RIPA buffer1x with 500 μl ice cold RIPA/high salt buffer2x with 500 μl ice cold LiCl wash buffer1x with 200 μl ice cold TE (quick wash and change to new tube)
?. [POOL AND SPLIT]
Transfer supernatant to a fresh tube.
Remember to save appropriate amount of the pool as input ( Usually 5-10% of the volume used for  the IPs)
?. [POOL AND SPLIT]
Combine all samples into a single tube; spin down at  for  at .
Centrifuge: 24000 33
4 °C
?. [LIBRARY PREPARATION]
Invitro Transcription (IVT)
?. [LIBRARY PREPARATION]
Prepare IVT mix in PCR tubes as follows:  AB1DNA from IP82NTP buffer mix (2x)103T7 RNA polymerase mix24Total volume20 μl
AB1DNA from IP82NTP buffer mix (2x)103T7 RNA polymerase mix24Total volume20 μl
It is advisable to take as much ChIP DNA as possible into the IVT reaction to maximize the library diversity
?. [LIBRARY PREPARATION]
Silane bead cleanup to isolate RNA (room temperature) Magnetize  of Silane beads per sample (will bind up to 3 μg RNA). Wash with  buffer RLT, magnetize, aspirate and resuspend beads in  buffer RLT.Add washed beads to sample, wait Add  100% EtOH (final: 0.6x), mix well, wait  (mix by pipetting at 2.5 min)Magnetize, wash twice with  70% EtOHAir-dry at RT for atleast  RNA eluted in  EBMeasure  on Qubit
15 µl
60 µl
60 µl
60 µl
20 µl
48 µl
200 µl
10 µl
1 µl
?. [IMMUNOPRECIPITATION]
Digest at for (native chromatin) or overnight (to reverse crosslink fixed chromatin).
63 °C
?. [IMMUNOPRECIPITATION]
Resuspend chromatin/antibody/beads in  of ChIP elution buffer and  of Proteinase K.
97.5 µl
2.5 µl
Remember to process the INPUT sample collected in Step 9.1, similar to the IPs from this step on.
?. [IMMUNOPRECIPITATION]
Elution from protein G beads and protein digestion
?. [LIBRARY PREPARATION]
RNA 3’ adapter ligation
?. [LIBRARY PREPARATION]
Set up the ligation rxn as follows per sample:  AB1RNA from IVT82RNA3 diluted adapter (10 μM)13RNA ligase buffer24T4 RNA ligase truncated15RNase OUT16Total Volume13  μl
AB1RNA from IVT82RNA3 diluted adapter (10 μM)13RNA ligase buffer24T4 RNA ligase truncated15RNase OUT16Total Volume13  μl
?. [LIBRARY PREPARATION]
Incubate at   for .
25 °C
?. [LIBRARY PREPARATION]
Reverse Transcription
?. [LIBRARY PREPARATION]
Move tube to ice and top up with the following components of the reverse transcription reaction:  AB1RNA from above132RT primer (20μM)43Anneanling buffer14Total Volume18 μlHeat to  for  and place on ice for
AB1RNA from above132RT primer (20μM)43Anneanling buffer14Total Volume18 μl
65 °C
?. [LIBRARY PREPARATION]
Then add :  AB12x First-Strand Reaction Mix202SIII/RNase OUT Enzyme Mix23Total Volume40 μlIncubate in a thermocycler with following program :   AB125°C10 min250°C50 min385°C5 min44°C∞
AB12x First-Strand Reaction Mix202SIII/RNase OUT Enzyme Mix23Total Volume40 μl
AB125°C10 min250°C50 min385°C5 min44°C∞
?. [LIBRARY PREPARATION]
Add Rnase H, DNase free and incubate  at .(this helps for accurate cDNA measurements)
1 µl
37 °C
?. [LIBRARY PREPARATION]
Library PCR
?. [LIBRARY PREPARATION]
And set it up with the following cycling conditions:  ABC1TemperatureDurationCycles295°C3 min1x395°C15 sec465°C30 sec4 x572°C30 sec695°C15 sec772°C60 sec10 x872°C5 min94°C∞1x
ABC1TemperatureDurationCycles295°C3 min1x395°C15 sec465°C30 sec4 x572°C30 sec695°C15 sec772°C60 sec10 x872°C5 min94°C∞1x
?. [LIBRARY PREPARATION]
SPRI bead cleanup to isolate DNA (room temperature)Add  of nuclease free water to the PCR mixture to make final volume .Add  SPRI beads (1x select >100 bp fragments)Mix by pipetting, wait Magnetize forWash 2x with 70% ethanol (magnetize 1 minute)Air-dry at RT for >=Elute DNA in  of elution buffer.Measure on Qubit.
We employed 1x to select for all >100bp fragments; no specific size selection.But one can also size select at this step.
50 µl
100 µl
100 µl
200 µl
20 µl
1 µl
?. [LIBRARY PREPARATION]
Adjust cDNA conc. for all samples to be  (or a minimum of 100 ng) in a total volume of with nuclease free water and set up following PCR reaction mix :  AB1Per rxn2cDNA + water22.530.5 μM PCR primer mix (10 μM)2.542x PCR master mix255Total Volume50 μl
150 ng
22.5 µl
AB1Per rxn2cDNA + water22.530.5 μM PCR primer mix (10 μM)2.542x PCR master mix255Total Volume50 μl
?. [LIBRARY PREPARATION]
SPRI bead cleanup to isolate DNA (room temperature)Add  of nuclease free water to the cDNA mixture to make final volume 1Add  SPRI beads (1x select >100 bp fragments)Mix by pipetting, wait Magnetize forWash 2x with  70% ethanol (magnetize 1 minute)Air-dry at RT for >=Elute DNA in of TE and measure conc. on Nanodrop.
59 µl
0 µl
100 µl
200 µl
24 µl
?. [LIBRARY PREPARATION]
Incubate samples at  overnight
37 °C
[roughly]
?. [IMMUNOPRECIPITATION]
SPRI bead cleanup to isolate DNA (room temperature)Using magnet, transfer  eluate to new wells. Add  SPRI beads (1x, select >100 bp fragments)Mix by pipetting, wait Magnetize for Wash 2x with 70% ethanol (magnetize 1 minute)Air-dry at RT for >=Elute DNA in  EB and measure  with Qubit
100 µl
100 µl
200 µl
12 µl
1 µl
There is no 'right' amount of ChIP DNA. Selective antibodies against low abundant targets will yield little DNA
?. [T7 ADAPTER LIGATION]
Mix and incubate with agitation at 800 rpm  at  for  and then at  overnight.
37 Room temperature
16 °C
The manufacturer's protocol for the end repair and blunt end ligation suggests 60 minutes incubation at room temperature.We have obtained satisfactory results with just 2 hours incubation at room temperature. The overnight incubation is therefore optional and was introduced more as a break point for the protocol.
?. [PREPARING LIBRARY FOR ILLUMINA]