TITLE: rev-ChIP
AUTHORS: Lorane Texari, Carlos Guzman, Sven Heinz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify">Understanding the precise regulation of transcriptional programs in human health and disease requires the accurate identification and characterization of genomic regulatory networks. Next-generation sequencing (NGS) technologies are powerful, and widely applied tools to map the in vivo genome-wide location of transcription factors (TFs), histone modifications, chromatin accessibility, and nascent transcription that make up these regulatory networks. While chromatin immunoprecipitation followed by sequencing (ChIP-seq) is one of the oldest, and most-utilized experimental techniques to study the location and abundance of TFs, experiments still frequently require optimization to reproducibly yield good data with high signal-to-noise ratios due to the massive variability between possible antibody-antigen combinations and commercial reagents. </div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">To overcome these obstacles, we systematically carried out well over 500 ChIP-seq experiments designed to test every aspect of typical ChIP-seq experiments and developed rev-ChIP, a novel ChIP-seq method that is optimized for scalability, robustness, low-input, speed, cost efficiency and data quality. We find that rev-ChIP can be scaled to work for cell numbers ranging from millions to under a thousand, and from a single sample to 500 samples a week in a non-automated fashion with minimal hands-on time. Additionally, rev-ChIP has been tested on a variety of sample types ranging from cell lines to sorted primary cells and solid tissues. </div><style>
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[STEPS]
?. [Lysis and Sonication]
Thaw cell pellet on ice and resuspend cells in  of lysis buffer.
500 µl
We do not recommend using  of lysis buffer or tip sonication when sonicating less than 500K cells. In this case we suggest using Covaris ( ) or PIXUL ( ).
?. [Lysis and Sonication]
Sonicate samples for 7 cycles.
This step is dependent on crosslinking method, and cell line or tissue type and should be optimized.
Double crosslinked DNA is harder to sonicate and requires more rounds of sonication.
Make sure not to oversonicate your samples and keep them constantly cold.
Chromatin size range of 200-500.
?. [Input Cleanup]
Take of each sample (for input) and put them into new PCR strip.
10 µl
?. [Input Cleanup]
Dilute lysis buffer (LB3 - add  10% Triton X-100, Metivier - add  Metivier Dilution Buffer, RIPA - None). If splitting lysates for IP, make sure diluted samples are well mixed. Split lysates as required if needed.
55 µl
750 µl
?. [Input Cleanup]
Add  of Elution Buffer (10mM Tris pH8, 0.5% SDS, 5mM EDTA, 280mM NaCl) +  RNase A to each input sample and incubate for  at .
68 µl
1 µl
?. [Input Cleanup]
Add 55 of Proteinase K to each input sample and incubate at  for  and then at  for .
1 µl
?. [Immunoprecipitation]
Prepare Dynabeads A/G: capture Dynabeads on magnet, remove supernatant and resuspend in equal volume of appropriate lysis buffer.
For LB3 use LB3 + 1/9th volume of 10% Triton X-100, for Metivier use an equal volume of Metivier Dilution Buffer and for RIPA use an equal volume of RIPA buffer.
?. [Immunoprecipitation]
Add the appropriate volume of dynabeads beads + antibody to each ChIP sample.
We recommend using  of antibody +  of dynabeads if using 100,000 cells or more. We recommend using  of antibody +  of dynabeads if using 10,000 cells or less.
?. [Immunoprecipitation]
Incubate IP overnight on wheel at  (rotating at 8rpm).
Optionally, you can incubate IP at  for  in most cases and for most antibodies for with minimal loss in data quality.
?. [Input Cleanup]
Create mastermix of 1 SpeedBeads + 20% PEG8000/1.5M NaCL (8.5% PEG, 1M NaCl), mix thoroughly, and add  of mastermix to each input sample.
2 µl
120 µl
122 µl
?. [Input Cleanup]
Incubate at RT for .
?. [Input Cleanup]
Wash 2x with  of 80% EtOH on magnet (move strip side to side 10x slow, 10x fast).
200 µl
We suggest using a repeater pipette here to speed things up.
If using 4 PCR strips or less we suggest vortexing beads at speed 8 for 10 seconds.
?. [Input Cleanup]
Air-dry until cracks appear on bead pellet.
Air-drying should take approximately  .
?. [Input Cleanup]
Elute in  TT (0.05% Tween 20, 10mM Tris pH 8.0, cold).
15 µl
We suggest using a repeater pipette here to speed things up.
Can store at  and stop for the day.
?. [Washes]
Add 100x PIC to WBI/III and TET ( PIC per   WBI/WBIII/TET). Do 3x washes with  WBI + PIC, 3x washes with  WBIII + PIC, and 2x washes with  cold TET + PIC.
10 µl
1 ml
180 µl
180 µl
185 µl
We suggest using a multichannel pipette here to speed things up.
The addition of PIC (Protease Inhibitor Cocktail) is not required, but highly recommended.
?. [Washes]
Resuspend beads in  of cold TT using repeater pipette.
25 µl
?. [Library Preparation]
Collect beads and take  of each input supernatant (1-2ul for 500K cells) that will be library prepped and add  of TT to each input taken.
2 µl
23 µl
?. [Library Preparation]
Create a mastermix of  of Enzyme Mix End Prep +  of End Prep Reaction Buffer per sample, mix well and add  of mastermix to each sample. Incubate for  at  and then  at .
1 µl
3 µl
5 µl
?. [Library Preparation]
Add  of Bioo ChIP Adaptors (10.625uM) to each sample.
1 µl
?. [Library Preparation]
Create a mastermix of  Ligation Master Mix +  of Ligation Enhancer per sample, mix well, and add  of mastermix to each sample and incubate for  at .
15 µl
0 µl
15 µl
?. [Library Preparation]
Create a STOP solution mastermix of 10% SDS + 0.5M EDTA +  water per sample and add  of mastermix to each sample.
4 µl
3 µl
20 µl
27 µl
?. [Library Preparation]
Add of 5M NaCl to each sample.
4 µl
We recommend using a multichannel to speed things up.
?. [Proteinase K and Reverse Crosslinking]
Add  of Proteinase K to each sample using multichannel and incubate for  at  and then  at .
1 µl
Alternatively, FA-fixed can be incubated for  at  .
Can leave at  overnight.
?. [Cleanup]
Create mastermix of  SpeedBeads +  20% PEG8000/1.5M NaCL (8.5% PEG, 1M NaCl), mix thoroughly, and add  of mastermix into new tube strips, then collect and transfer the supernatant of samples into these new tubestrips with  of speedbeads + peg.
2 µl
61 µl
63 µl
63 µl
DO NOT MIX PROTEIN A/G BEADS WITH SPEEDBEADS
?. [Cleanup]
Incubate at RT for .
?. [Cleanup]
Wash 2x with  of 80% EtOH on magnet (move strip side to side 10x slow, 10x fast).
200 µl
We recommend using a repeater pipette to speed things up.
?. [Cleanup]
Air-dry until cracks appear on bead pellet.
Air-drying should take approximately  .
?. [Cleanup]
Elute in  TT (0.05% Tween 20, 10mM Tris pH 8.0, cold). Collect beads on magnet and transfer supernatant into new PCR strips.
25 µl
We recommend using a multichannel to speed things up.
?. [Library Prep Amplification PCR]
Do PCR.  ABC1Mastermix Library PCR225.5ul MM + 24.5ul sample1x20x3Sample24.54(Blue Cap) NEBNext Ultra II Q5 2x MM255005100uM Solexa 1GA0.2556100uM Solexa 1GB0.255  ABC1PCR Program298℃30 seconds3R98℃10 seconds460℃15 seconds572℃30 seconds672℃1 minute74℃ON
?. [Final Cleanup]
Make mastermix of  SpeedBeads +  of per sample and add  of mastermix to each sample.
2 µl
38 µl
40 µl
?. [Final Cleanup]
Incubate at RT for .
?. [Final Cleanup]
Wash 2x with  of 80% EtOH on magnet (move strip side to side 10x slow, 10x fast).
200 µl
We recommend using a repeater pipette to speed things up.
?. [Final Cleanup]
Air-dry until cracks appear on bead pellet.
Air-drying should take approximately  .
?. [Final Cleanup]
Elute in  of TT.
20 µl
?. [Qubit]
Qubit library prep using HS DNA buffer and standards.