title: GSM9375705: Rad21_CD71_Ins_aPonly_rep1; Mus musculus; ChIP-Seq
library_construction_protocol: CTCF Chromatin immunoprecipitation (ChIP) was performed on 1 x 10^7 Ter119+ erythroid cells using a ChIP Assay Kit (Millipore: 17-295) according to the manufacturer's instructions. Cells were crosslinked by a single 10 min 1% formaldehyde fixation. Chromatin fragmentation was performed with the Bioruptor Pico sonicator (Diagenode) for a total sonication time of 4 min (8 cycles) at 4°C to obtain an average fragment size between 200 and 400 bp. Immunoprecipitation was performed overnight at 4 °C with an anti-CTCF antibody (10 µl 07-729, lot: 2836926; Millipore). Rad21 ChIP performed on 1-3 x10^6 CD71+ cells. Cells were double cross-linked using disuccinimidyl glutarate for 30 mins (DSG, Sigma) and 1% formaldehyde (Sigma) for 30 mins. Chromatin fragmentation was performed with a Covaris ME220  (600secs, 75 power, 25% Duty Factor, 1000 cycles per burst at 8°C to obtain an average fragment size between 200 and 400 bp. Immunoprecipitation was performed overnight at 4 °C with an anti-Rad21 antibody (10 µl ab154769, lot: 11035529-12; abcam). ChIP DNA was collected using blocked Protein A/G Dynabeads (Invitrogen; 100-01D/100-03D). CTCF ChIP-seq: DNA was purified by phenol-chloroform extraction with PhaseLock tubes (5Prime) and ethanol precipitation with NaOAc, and 2 µl GlycoBlue (Invitrogen). ChIP enrichment was determined by RT-qPCR. The NEBNext Ultra II DNA Library Prep Kit (New England Biolabs: NEB: 7645S/L) was used to prepare indexed sequencing libraries following the manufacturer supplied protocol. PCR amplification was performed for 7-11 cycles (depending on input DNA concentration) using NEBNext Mulitplex Oligos (New England Biolabs: E7335S/L and E7500S/L). Rad21 ChIP-seq: DNA was purified following RNase and Proteinase K treatment using the ChIP Clean and Concentrate kit (Zymo; D5205). Libraries were prepared using NEBNext Ultra II DNA Library Prep Kit and NEB index primers, with PCR amplication for 10 cycles.