title: GSM2973930: Neu-D1_HiC_rep1; Homo sapiens; Hi-C
library_construction_protocol: For RNA-seq, total mRNA was isolated from Neu-D1 and Neu-D3 cells using the Qiagen RNeasy kit and processed for Illumina library preparation and sequencing. For ChIP-seq of lamin B1, cells at each differentiation time point were fixed with 1% formaldehyde for 10 min, lysed for 10 min in ChIP lysis buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS, protease inhibitors and 1 mM PMSF) and sonicated 4 times 10 min in a Bioruptor (Diagenode) to generate 200 base-pair DNA fragments. After sedimentation, the supernatant was collected and diluted 10-fold in RIPA (140 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, protease inhibitors, 1 mM PMSF). Chromatin was incubated overnight at 4oC with the anti-lamin B1 antibody coupled to Dynabeads Protein A/G (Invitrogen). ChIP samples were washed 4 times in ice-cold RIPA, crosslinks reversed and DNA eluted for 6 h at 68oC in 50 mM NaCl, 20 mM Tris-HCl pH 7.5, 5 mM EDTA, 1% SDS and 50 ng/µl Proteinase K. DNA was purified using phenol-chloroform isoamylalcohol and dissolved in H2O prior to processing for Illumina library preparation and sequencing. For Hi-C, 25 million cells per time point and differentiation replicate were cross-linked with fresh 1% formaldehyde (Pierce) for 12 min at room temperature on a rocker. Excess formaldehyde was quenched with 0.125 M glycine for 5 min and cells washed 3 times 5 min in ice-cold PBS. Crosslinked cells were lysed in lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630 and protease inhibitors) on ice for 15 min and homogenised by Douncing with a B-pestle (tight) in lysis buffer until nuclei were released, as assessed by microscopy. The lysate was sedimented, the pellet was resuspended in NEBuffer 2 (10 mM Tris-HCl pH 7.9, 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol) containing 0.1% SDS, and incubated at 65oC for 10 min. SDS was quenched on ice with 1% Triton X-100 and the sample digested with HindIII overnight at 37oC. Digested fragments were labelled with biotin-14-dCTP and blunt-end ligated. Crosslinks were reversed at 65oC overnight, the ligated DNA was purified by phenol-chloroform:isoamylalcohol (Ambion), desalted and RNAse-treated. DNA was sheared to ~100-300 base-pairs by Covaris sonication and size-fractionated on AMpure XP magnetic beads (Beckman Coulter). Biotinylated DNA fragments were pulled-down with Streptavidin beads and processed for Illumina adapter ligation and library preparation. Illumina sequencing libraries were generated as per Illumina protocols.
attributes.tag: GEO Accession
attributes.value: GSM2973930