Dec 03, 2018

Public workspacerev-ChIP V.2

DOI
dx.doi.org/10.17504/protocols.io.vrbe52n
  • 1University of California, San Diego
Carlos Guzman
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DOI: dx.doi.org/10.17504/protocols.io.vrbe52n
Protocol Citation: Lorane Texari, Carlos Guzman, Sven Heinz 2018. rev-ChIP. protocols.io https://dx.doi.org/10.17504/protocols.io.vrbe52n
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol in our group and it is working. Developments and edits occur as needed.
Created: November 20, 2018
Last Modified: December 03, 2018
Protocol Integer ID: 17923
Keywords: ChIP-seq
Abstract
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.

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.
Lysis and Sonication
Lysis and Sonication
Thaw cell pellet on ice and resuspend cells in Amount500 µL of lysis buffer.


Note
We do not recommend using Amount500 µL of lysis buffer or tip sonication when sonicating less than 500K cells. In this case we suggest using Covaris (Amount130 µL ) or PIXUL (Amount60 µL ).




Sonicate samples for 7 cycles.

Note
This step is dependent on crosslinking method, and cell line or tissue type and should be optimized.

Note
Double crosslinked DNA is harder to sonicate and requires more rounds of sonication.

Note
Make sure not to oversonicate your samples and keep them constantly cold.

Expected result
Chromatin size range of 200-500.

Input Cleanup
Input Cleanup
Take Amount10 µL of each sample (for input) and put them into new PCR strip.
Dilute lysis buffer (LB3 - add Amount55 µL 10% Triton X-100, Metivier - add Amount750 µL Metivier Dilution Buffer, RIPA - None). If splitting lysates for IP, make sure diluted samples are well mixed. Split lysates as required if needed.

Add Amount68 µL of Elution Buffer (10mM Tris pH8, 0.5% SDS, 5mM EDTA, 280mM NaCl) + Amount1 µL RNase A to each input sample and incubate for Duration00:15:00 at Temperature37 °C .
Add Amount1 µL 55 of Proteinase K to each input sample and incubate at Temperature55 °C for Duration01:00:00 and then at Temperature65 °C for Duration00:30:00 .
Immunoprecipitation
Immunoprecipitation
Prepare Dynabeads A/G: capture Dynabeads on magnet, remove supernatant and resuspend in equal volume of appropriate lysis buffer.
Note
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.

Add the appropriate volume of dynabeads beads + antibody to each ChIP sample.
Note
We recommend using Amount1 µg of antibody + Amount10 µL of dynabeads if using 100,000 cells or more.

We recommend using Amount0.1 µg of antibody + Amount1 µL of dynabeads if using 10,000 cells or less.

Incubate IP overnight on wheel at Temperature4 °C (rotating at 8rpm).
Note
Optionally, you can incubate IP at Temperature4 °C for Duration01:00:00 in most cases and for most antibodies for with minimal loss in data quality.


Input Cleanup
Input Cleanup
Create mastermix of Amount2 µL 1 SpeedBeads + Amount120 µL 20% PEG8000/1.5M NaCL (8.5% PEG, 1M NaCl), mix thoroughly, and add Amount122 µL of mastermix to each input sample.
Incubate at RT for Duration00:10:00 .
Wash 2x with Amount200 µL of 80% EtOH on magnet (move strip side to side 10x slow, 10x fast).

Note
We suggest using a repeater pipette here to speed things up.

Note
If using 4 PCR strips or less we suggest vortexing beads at speed 8 for 10 seconds.

Air-dry until cracks appear on bead pellet.

Note
Air-drying should take approximately Duration00:14:00 .


Elute in Amount15 µL TT (0.05% Tween 20, 10mM Tris pH 8.0, cold).

Note
We suggest using a repeater pipette here to speed things up.

Note
Can store at Temperature-20 °C and stop for the day.



Washes
Washes
Add 100x PIC to WBI/III and TET (Amount10 µL PIC per Amount1 mL WBI/WBIII/TET). Do 3x washes with Amount180 µL WBI + PIC, 3x washes with Amount180 µL WBIII + PIC, and 2x washes with Amount185 µL cold TET + PIC.
Note
We suggest using a multichannel pipette here to speed things up.

Note
The addition of PIC (Protease Inhibitor Cocktail) is not required, but highly recommended.

Resuspend beads in Amount25 µL of cold TT using repeater pipette.

Library Preparation
Library Preparation
Collect beads and take Amount2 µL of each input supernatant (1-2ul for 500K cells) that will be library prepped and add Amount23 µL of TT to each input taken.


Create a mastermix of Amount1.5 µL of Enzyme Mix End Prep + Amount3.5 µL of End Prep Reaction Buffer per sample, mix well and add Amount5 µL of mastermix to each sample. Incubate for Duration00:30:00 at Temperature20 °C and then Duration00:30:00 at Temperature65 °C .

Add Amount1 µL of Bioo ChIP Adaptors (10.625uM) to each sample.
Create a mastermix of Amount15 µL Ligation Master Mix + Amount0.5 µL of Ligation Enhancer per sample, mix well, and add Amount15.5 µL of mastermix to each sample and incubate for Duration00:15:00 at Temperature20 °C .

Create a STOP solution mastermix of Amount4 µL 10% SDS + Amount3 µL 0.5M EDTA + Amount20 µL water per sample and add Amount27 µL of mastermix to each sample.

Add Amount4.5 µL of 5M NaCl to each sample.
Note
We recommend using a multichannel to speed things up.


Proteinase K and Reverse Crosslinking
Proteinase K and Reverse Crosslinking
Add Amount1 µL of Proteinase K to each sample using multichannel and incubate for Duration01:00:00 at Temperature55 °C and then Duration00:30:00 at Temperature65 °C .
Note
Alternatively, FA-fixed can be incubated for Duration00:30:00 at Temperature55 °C .

Note
Can leave at Temperature4 °C overnight.


Cleanup
Cleanup
Create mastermix of Amount2 µL SpeedBeads + Amount61 µL 20% PEG8000/1.5M NaCL (8.5% PEG, 1M NaCl), mix thoroughly, and add Amount63 µL of mastermix into new tube strips, then collect and transfer the supernatant of samples into these new tubestrips with Amount63 µL of speedbeads + peg.

Note
DO NOT MIX PROTEIN A/G BEADS WITH SPEEDBEADS


Incubate at RT for Duration00:10:00 .
Wash 2x with Amount200 µL of 80% EtOH on magnet (move strip side to side 10x slow, 10x fast).

Note
We recommend using a repeater pipette to speed things up.

Air-dry until cracks appear on bead pellet.
Note
Air-drying should take approximately Duration00:14:00 .

Elute in Amount25 µL TT (0.05% Tween 20, 10mM Tris pH 8.0, cold). Collect beads on magnet and transfer supernatant into new PCR strips.
Note
We recommend using a multichannel to speed things up.


Library Prep Amplification PCR
Library Prep Amplification PCR
Do PCR.
Mastermix Library PCR
25.5ul MM + 24.5ul sample1x20x
Sample24.5
(Blue Cap) NEBNext Ultra II Q5 2x MM25500
100uM Solexa 1GA0.255
100uM Solexa 1GB0.255

PCR Program

98℃30 seconds
R98℃10 seconds
60℃15 seconds
72℃30 seconds

72℃1 minute

4℃ON

Final Cleanup
Final Cleanup
Make mastermix of Amount2 µL SpeedBeads + Amount38.5 µL of per sample and add Amount40.5 µL of mastermix to each sample.
Incubate at RT for Duration00:10:00 .
Wash 2x with Amount200 µL of 80% EtOH on magnet (move strip side to side 10x slow, 10x fast).
Note
We recommend using a repeater pipette to speed things up.

Air-dry until cracks appear on bead pellet.
Note
Air-drying should take approximately Duration00:14:00 .

Elute in Amount20 µL of TT.
Qubit
Qubit
Qubit library prep using HS DNA buffer and standards.