GSM3763122: Scid.adh.2c2_sgCont_RNA-rep1; Mus musculus; RNA-Seq
ChIP-seq; Ten million of ILC2/b6 or Schd.adh.2c2 cells were fixed with 1 mg/ml DSG (Thermo Scientific) in PBS for 30 min at RT followed by additional 10 min with addition of formaldehyde up to 1%.  The reaction was quenched by addition of 1/10 volume of 0.125M glycine and the cells were washed with HBSS (Gibco).  Pelleted nucleus were dissolved in lysis buffer (0.5% SDS, 10 mM EDTA, 0.5 mM EGTA, 50 mM Tris-HCl (pH 8) and PIC) and sonicated on a Bioruptor (Diagenode) for 18 cycles of 30sec sonication followed by 30sec rest, with max power.  Five μg per 107 cells of anti-Bcl11b Abs (a mixture of A300-383A (Bethyl), A300-385A (Bethyl), ab18465 (Abcam) and 12120 (CST)), anti-Runx3 (kindly provided from Dr. Ditsa Levanon and Dr. Yoram Groner, Weizmann Institute of Science), anti-GATA3 (a mixture of sc-268, Santa Cruz and MAB26051, R&D) or anti-Runx1 (ab23980, Abcam) were hybridized to Dynabeads anti-Rabbit, Dynabeads anti-Mouse or Dynabeads Protein A/G (Invitrogen) and added to the diluted chromatin complex.  They were incubated over night at 4°C, then washed and eluted for 6hr at 65°C in ChIP elution buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA 50 mM NaCl, 1% SDS, and 50 μg/ml proteinase K).  Precipitated chromatin fragments were cleaned up using Zymo ChIP DNA Clean & Concentrator.  ChIP-seq libraries were constructed using NEBNext ChIP-Seq Library Preparation Kit (E6240, NEB) and sequenced on Illumina HiSeq2500 in single read mode with the read length of 50 nt. RNA-seq; Total RNA was isolated from 300,000 cells using an RNAeasy MicroKit (Qiagen).  Libraries were constructed using NEBNext Ultra RNA Library Prep Kit for Illumina (E7530, NEB) from ~1 μg of total RNA following manufacturer's instructions.  Libraries were sequenced on Illumina HiSeq2500 in single read mode with the read length of 50 nt.