title: GSM3903798: HiChIP-Gata1-EryD-Rep2; Mus musculus; OTHER
library_construction_protocol: HiChIP was performed as previously described (Mumbach et al 2016) using the Gata1 antibody (ab11852, Abcam). In brief, 15 million of either peripheral blood cells of E10.5 mouse embryos or single cells of E14.5 fetal liver were cross-linked with 1% v/v formaldehyde at room temperature for 10 minutes, and were quenched with 0.125 M Glycine for 5minutes. Crosslinked cells were lysed in cold Hi-C lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2% NP40, 1x Roche protease inhibitors) and followed by 1 U/ul MboI (NEB) digestion at 37oC for 6 h, fill-in of overhangs with biotin-dATP (Thermo Fisher) at 37oC for 1.5 h, and ligation with 4 U/ul T4 DNA Ligase (NEB) at room temperature for 6 h. The nuclear pellet was sheared using a Covaris E220 with following parameters: Duty Factor = 5.0, Peak Power = 140, Cycle/Burst = 200, Time = 240 seconds. Chromatin extracts were immunoprecipitated with Gata1 antibodies overnight at 4oC, and then incubated with protein A Dynabeads (Invitrogen) for another 4 hours. ChIP complexes were eluted and cross-linking reversed by heating at 65oC overnight. DNA was extracted with DNA Clean and Concentrator columns (Zymo Research). ATAC-seq was performed as described (Buenrostro et al., 2013) on FACS isolated EryP and EryD cells. 2.5 x 104 cells were used per sample. 2.5 l Tn5 Transposases (Illuminar) was used to make the transposition reaction. Libraries were generated using Ad1_noMX and Ad2.1-2.4 barcoded primers (Buenrostro et al., 2013) and were amplified for 6-9 total cycles. All libraries were sequenced on Illumina HiSeq2000 with 100 bp paired-end reads. HiChIP was performed as previously described (Mumbach et al., 2016) using the Gata1 antibody (ab11852, Abcam). In brief, 15 million of either peripheral blood cells of E10.5 mouse embryos or single cells of E14.5 fetal liver were cross-linked with 1% v/v formaldehyde at room temperature for 10 minutes, and were quenched with 0.125 M Glycine for 5minutes. Crosslinked cells were lysed in cold Hi-C lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2% NP40, 1x Roche protease inhibitors) and followed by 1 U/l MboI (NEB) digestion at 37oC for 6 h, fill-in of overhangs with biotin-dATP (Thermo Fisher) at 37oC for 1.5 h, and ligation with 4 U/l T4 DNA Ligase (NEB) at room temperature for 6 h. The nuclear pellet was sheared using a Covaris E220 with following parameters: Duty Factor = 5.0, Peak Power = 140, Cycle/Burst = 200, Time = 240 seconds. Chromatin extracts were immunoprecipitated with Gata1 antibodies overnight at 4oC, and then incubated with protein A Dynabeads (Invitrogen) for another 4 hours. ChIP complexes were eluted and cross-linking reversed by heating at 65oC overnight. DNA was extracted with DNA Clean and Concentrator columns (Zymo Research).   For library preparation, Biotin labeled post-ChIP DNA was captured by 5 l Streptavidin C-1 beads (Thermo Fisher), and followed by Tn5 treatment. The amount of Tn5 used and the number of PCR cycles performed was based on the post-ChIP Qubit amounts, as previously described (Mumbach et al., 2016). HiChIP libraries were size-selected for 300-800bp fragments. All libraries were sequenced on the Illumina Nextseq 500 to an average depth of 200 million total reads.