GSM5851497: ES_ChIL100C_S2P_1; Mus musculus; OTHER
ChIL-seq was performed following a modified protocol of the original ChIL-seq (PMID: 30532068). For ES cells, 100 or 1000 cells were plated on gelatin coated 96-well plates and let to attach for 16h in culture. Cells were then fixed with 1% formaldehyde in DMEM for 5 min and permeabilized with 1% Triton X-100 in PBS for 20min. Embryos were fixed using the same procedure. Samples were incubated in 2% BSA in PBS with anti-Pol II Ser5P (Abcam, ab5131) or anti-Pol II Ser2P (Abcam, ab5095) at 2μg/ml for 6h at room temperature, washed three times in 0.5% BSA in PBS and then incubated at 4 °C overnight in 2% BSA in PBS containing 500mM NaCl and the ChIL probe at 2μg/ml. After washing in PBS for ES-cells or PBS containing 0.1% BSA for embryos, samples were incubated in 50 µl Tn5 solution (50mM HEPES-KOH, pH 7.2, 0.1M NaCl, 0.1mM EDTA, 1mM dithiothreitol, 0.1% Triton X-100 and 10% glycerol) containing 0.5µl HActive™ Tn5 transposase (Glow Biologics, GBRP-TN5) for 10min at room temperature. Tn5-MEDS-B oligo (2 µM) was then added at 0.5 µl per well in 1× dialysis buffer (50mM HEPES-KOH, pH 7.2, 0.1M NaCl, 0.1mM EDTA, 1mM dithiothreitol, 0.1% Triton X-100 and 10% glycerol) and incubated for 60min at room temperature. Samples were then washed in 1× dialysis buffer and three times in PBS for ES-cells or PBS containing 0.1% BSA for embryos, incubated in 100 µl 1× TAPS-DMF buffer (10mM TAPS-NaOH, pH 8.5, 5mM MgCl2 and 10% N,N-dimethylformamide) for 1h at 37 °C, and washed at three times in PBS for ES-cells or PBS containing 0.1% BSA for embryos. To stop the Tn5 reaction, cells were incubated in 100 µl 0.2% SDS for 10min at room temperature or embryos were incubated in 0.1% BSA/PBS containing 50mM EDTA at 50 °C for 30 min. After washing at three times in PBS for ES-cells or PBS containing 0.1% BSA for embryos, samples were incubated in 50 µl reaction mixture (200U T4 DNA ligase, NEB, and 1.5U T4 DNA polymerase, NEB, in 1× T4 DNA ligase reaction buffer containing 0.1 mM dNTP) for 30min at room temperature. After discarding the solution, for ES cells, 100µl 0.2% SDS were added to remove DNA-bound enzymes, followed by incubation for 10min at room temperature and three washes in PBS. For embryos, 0.1% BSA/PBS containing 50mM EDTA was added for 30 min at 50 °C, and then washed at three times in 0.1% BSA/PBS. In situ transcription was then carried out using T7 RNA polymerase (800U per well; NEB) for 20h at 37 °C. The samples were treated with DNase I (2.5U per well; Thermo) for 30min at 37 °C and RNA was extracted using a Qiagen MinElute Cleanup kit. Purified RNA was reverse-transcribed with SMARTScribe reverse transcriptase (Takara) following the manufacturer's instructions and the resulting cDNA was amplified by Prime star GXL (Takara) by 14 cycles for 1000 cells with anti-Pol II Ser5P antibody, 15 cycles for 1000 cells with anti-Pol II Ser2P antibody, 16 cycles for 100 cells or embryos with anti-Ser5P antibody, or 17 cycles for 100 cells or embryos with anti-Ser2P antibody. After amplification, small fragments (<150bp) were removed using Gene read Size selection kit (Qiagen) and large size of fragments (>600bp) were removed using AMPure XP beads (Beckman Coulter). The amplified DNA was purified using AMPure XP beads and the quality of the libraries was assessed using a Bioanalyzer 2100 (Agilent Technologies). Samples were paired-end sequenced at PE150 on an Illumina NovaSeq 6000 platform