title: GSM7831961: ESC_X_NANOG_RA_2; Mus musculus; ChIP-Seq
library_construction_protocol: For crosslinked MINUTE-ChIP, MINUTE-ChIP (PMID: 31533047) was adapted as follows: cells were harvested via trypsinization and fixed for 10 min at RT with 1 % Formaldehyde. Subsequently, the formaldehyde was quenched using 0.75 M Tris-HCl (ph 7.8) for 10 min at RT. The cell  were then pelleted by centrifugation at 800 g, 4 °C for 5 min, and washed twice using cold PBS. During the final PBS wash, 1e6 cells were pelleted per aliquot.  Pellets were flash frozen and stored at -80 °C. Flash frozen pellets of 2e6 cells were resuspended in 50uL of sonication buffer (50mM Tris HCl, 0.5% SDS, 1x PI), transferred to 0.5mL bioruptor tubes and lysed on ice for 10 mins. Samples were then sonicated for 40 mins, high power, 30 sec on/off  cycles on Biruptor sonicator ( 4 iterations of 10 mins each and between each  iteration, tubes were briefly vortexed and spun down).  The sheared cell suspension was spun down at 800g, 4 °C for 5 min and the supernatant was transferred to a fresh tube and topped up with 50uL of sonication dilution buffer (50mM Tris HCl, 1% Triton X-100).  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. 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). 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 3 μl Nanog antibody (ab80892). 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 crosslinked ChIP DNA was treated with Protinase K at 65C for 4h and 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.