alias: GSM2627208
title: GSM2627208: Control RNA-seq rep1; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was prepared using TRIzol reagent (Thermal Fisher Scientific, cat. no. 15596018). In situ Hi-C experiments were performed in RWPE-1 cells following the original protocol by Rao et al with minor modifications. Hi-C was performed in duplicate and 5 x 106 cells were used for each experiment. 100U of MboI restriction enzyme (NEB, R0147) was used to digest chromatin. For ligation, 2000U T4 DNA Ligase (NEB, M0202) was added and incubated at room temperature for 4 hours with slow rotation. Hi-C material was sheared to a size of 300-500bp using a Covaris instrument (Covaris S2, Woburn, MA). Biotin-tagged DNA was pulled down using Dynabeads MyOne Streptavidin C1 beads (Life technologies, 65002) with 2X Binding Buffer (2X BB: 10mM Tris-HCl (pH 7.5), 1nM EDTA, 2M NaCl). Hi-C library was amplified with 14 cycles of PCR using Illumina primers. Each library was sequenced with 75bp paired-end for ~500M read pairs using Illumina HiSeq 2000. The RNA-seq libraries were made using KAPA stranded mRNA-seq kits (KAPK Biosystems, cat. no. kk8421) according to manufacturer's instructions In situ HiC experiments were performed in RWPE-1 cells following the original protocol by Rao et al with minor modifications. HiC was performed in duplicate and 5 x 106 cells were used for each experiment. 100U of MboI restriction enzyme (NEB, R0147) was used to digest chromatin. For ligation, 2000U T4 DNA Ligase (NEB, M0202) was added and incubated at room temperature for 4 hours with slow rotation. HiC material was sheared to a size of 300-500bp using a Covaris instrument (Covaris S2, Woburn, MA). Biotin-tagged DNA was pulled down using Dynabeads MyOne Streptavidin C1 beads (Life technologies, 65002) with 2X Binding Buffer (2X BB: 10mM Tris-HCl (pH 7.5), 1nM EDTA, 2M NaCl). HiC library was amplified with 14 cycles of PCR using Illumina primers.
identifiers.namespace: GEO
identifiers.id: GSM2627208