GSM5331858: CDNI RNA-seq rep2; Homo sapiens; RNA-Seq
Biochemical fractionation was performed on 108 TIG-1 fibroblasts harvested with trypsin and thoroughly washed with ice cold PBS. Buffers at all steps contained protease inhibitors (Roche) and RNase inhibitor where appropriate (RNasin, Promega). First, nuclei were isolated by thoroughly resuspension in 1 ml Isotonic Lysis Buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 3 mM MgCl2, 10% glycerol, 0.5% NP-40, 0.5 mM PMSF) and incubating for 15 min on ice with gentle vortexing every 5 minutes. Nuclei were pelleted by centrifugation at 4°C for 3 min at 1000 g. The cytosolic supernatant was removed and permeabilized nuclei were washed twice in Isotonic Lysis Buffer lacking NP-40 with 5 min incubations on ice and gentle vortexing between and recovered by centrifugation as above. Loosely bound nucleoplasmic proteins and RNAs were then solubilized by suspension in 1 ml ice cold modified urea/NP-40 Buffer (1 M urea, 1% NP-40, 0.5 mM PMSF) (Bhatt et al., 2012). Nuclei were incubated for 15 minutes with vigorous vortexing every 5 minutes. The insoluble nuclear material was then pelleted by centrifugation at 4°C for 10 min at 10,000 g. The supernatant was collected as the nuclear soluble (NS) fraction. The insoluble material was resuspended in urea/NP-40 buffer and washed after another incubation of 15 minutes on ice with additional vortexing. The insoluble material was then extracted with 0.5 ml Ammonium Sulfate Extraction Buffer (10 mM Tris-HCl pH 7.5, 250 mM ammonium sulfate, 300 mM sucrose, 3 mM MgCl2, 0.5% Triton X-100, 0.5 mM PMSF) for 10 minutes on ice with vigorous vortexing to disrupt the insoluble pellet. Extracted insoluble material was then pelleted at 4°C for 10 min at 10,000 g. The supernatant was collected and the insoluble pellet was again washed as above in Ammonium Sulfate Extraction Buffer. The insoluble pellet was then washed wih 0.5 ml DNase Digestion Buffer (40 mM Trsi-HCl pH 7.9, 10 mM NaCl, 6 mM MgCl2, 1 mM CaCl2, 0.05% NP-40, 0.5 mM PMSF). Insoluble material was again suspended in 0.5 ml DNase Digestion Buffer before addition of 10 µl DNase I (Roche). DNA was digested for 60 minutes at 37°C. Samples were extensively vortexed before an additional 10 µl DNase I (Turbo DNase, Ambion) was added and samples were again incubated for 30 minutes at 37°C. The insoluble nuclear material was then pelleted by centrifugation at 4°C for 10 min at 10,000 g. Supernatants from this step and the ammonium sulfate extraction were combined as the chromatin-dependent fractions. Insoluble material was subjected to an additional high salt wash by being thoroughly resuspended in 0.5ml High Salt Wash Buffer (10 mM Tris-HCl pH 7.5, 10% glycerol, 2 M NaCl, 3 mM MgCl2, 0.05% NP-40, 0.5 mM PMSF). Samples were vortexed and incubated 5 min on ice. The final insoluble scaffold material was then pelleted by centrifugation at 4°C for 10 min at 10,000 g. RNA was recovered by the addition of Trizol (Invitrogen) and EDTA (10 mM final), following ethanol precipitation where necessary. Samples were heated to 65°C for 15 minutes with intermittent vortexing before RNA isolation according to manufacturer recommendations. Trizol isolated RNA was further cleaned up using RNAEasy columns (Qiagen). Libraries were prepared using a stand specific kit RNA-seq kit with ribosome depletion (KAPA Biosystems) and NEBNext Adapters and Multiplex Oligos for Illumina sequencing.