GSM3899122: ESC IN (0:100 L:H); Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA
Attributes by original data submitter
Sample
source_name
Embryonic Stem Cell
background
Rw4 (129X1/SvJ)
condition
Serum
antibody
Input
mixing ratio l
h: 0:100
supplementary file scaling
1x genome coverage
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
MINUTE-ChIP and library preparation protocol is based on the Mint-ChIP protocol developed by the Bernstein lab (van Galen et al., 2016), with modifications as follows: 1x106 cells were harvested for each growth condition, washed twice with PBS and cell pellets were flash frozen at -80°C prior to use. Cells were resuspended in 50 μl PBS, lysed and digested to mono- to tri-nucleosomes fragments by adding 50 μl of 2x Lysis buffer (100 mM Tris-HCL [pH 8.0], 0.2% Triton X-100, 0.1% sodium deoxycholate, 10 mM CaCl2 and 1x PIC) containing 2U/μl of micrococcal nuclease (New England BioLabs, M0247S) and incubating on ice for 20 min and then 37°C for 10 min. Double-stranded DNA adaptors for barcoding and T7 amplification were generated by slow annealing of complementary single-stranded oligos (for sequences refer to Supplementary Table 1), where the UMI bases were randomized. While resulting dsDNA adaptors may contain mismatched bases within the UMI, only the bottom strand UMI is amplified and sequenced. For each sample, 40 μl of the whole cell lysate containing the digested chromatin was taken forward into an overnight blunt end ligation reaction (End-It DNA repair kit and Fast-Link DNA ligation kit, Epicentre) with double stranded DNA adapters at 16°C. As in the original Mint-ChIP design, adaptors carried a partial SBS3 for Illumina sequencing flanked by a T7 RNA Polymerase for linear amplification. Between the SBS3 sequence and a 8bp sample barcode at the 3' end, a 6bp randomized sequence was introduced, serving as a unique molecular identifier (UMI) (Figure 1a). UMI and sample barcode are ligated 5' to the chromatin fragment and constitute the first 14 bases of read 1. The 4096 possible UMIs provide sufficient diversity to distinguish if two reads mapping to the exact same genomic location arose from a PCR amplification artifact or are indeed unique molecule. The adapter concentration was optimized to 2.5 μM / reaction to reduce adapter dimers. The ligation reaction was terminated with a lysis dilution buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% Triton X-100, 50 mM EGTA, 50 mM EDTA, 0.1% sodium deoxycholate and 1x PIC) and barcoded samples were combined into a single pool, spun down at 24,000 r.p.m. for 10 min at 4°C. 50μl Protein A/G magnetic beads (BioRad) were washed twice with PBS-T (PBS+ 0.1% Tween 20) and coupled to one of the following antibodies in the same buffer for 1 hr at room temperature with rotation: 3 μl H3 (Active motif 39763), 5 μl H3K4me3 (Millipore 04-745), 5 μl H3K27me1 (Cell signaling 5326S), 5 μl H3K27me2 (Cell signalling D18C8), 5 μl H3K27me3 (Cell signaling 9733 (rab mAb) or Diagenode C15410195 (rab pAb) or Millipore 07-449 (rab pAb)). Beads were then washed quickly with RIPA buffer. 200-400 μl of the cleared lysate pool was added to the pre-coupled magnetic beads and parallel ChIP assays were incubated further for 4 h at 4°C with rotation. 5% of the above volume was saved as the input pool and processed through the remaining protocol in a manner similar to the IPs. Next, the beads were washed (RIPA, RIPA high salt, LiCl and TE buffer) resuspended in ChIP elution buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 0.1% SDS, and 300 mM NaCl) containing 0.25 mg/mL Proteinase K and eluted at 63°C for 1 h. The native ChIP DNA was isolated using 1x SPRI beads (Beckman Coulter), retaining predominantly fragments > 100bp, and set up in an overnight in vitro transcription reaction (HiScribe T7 Quick High Yield RNA Synthesis kit, New England BioLabs). The resulting RNA was purified using Silane beads (Life Technologies) and an RNA 3' adapter was ligated onto it using T4 RNA ligase, truncated (New England BioLabs) for 1 h at 25°C. The mixture was subsequently supplemented with components of the reverse transcription reaction (SuperScript III First-Strand Synthesis SuperMix, Life Technologies) to produce cDNA, primed using the ligated 3' adapter. Final libraries for each ChIP were produced using 150-200 ng of purified cDNA in a PCR reaction (High-Fidelity 2x master mix, New England BioLabs) for 8 cycles with 0.2 μM primers that carried a second 8bp barcode sequence. Quality assessment and concentration estimation of the purified DNA was done using the Qubit (Life Technologies) and BioAnalyzer (Agilent). Each library was then diluted to 4 nM and combined into a single pool before sequencing on the Illumina NextSeq500 platform.