GSM5331861: control SAR-seq rep1; Homo sapiens; OTHER
Scaffold attached DNA was isolated from 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 resuspending 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 5 min. Nuclei were pelleted by centrifugation at 4°C for 3 min at 800 g. The cytosolic supernatant was removed and permeabilized nuclei were washed once in Isotonic Lysis Buffer lacking NP-40. Nuclei were then incubated for 5 minutes at 37°C in 0.5 ml Isotonic Lysis Buffer lacking NP-40 with the addition of RNase inhibitor or RNase A (Thermo Fisher, 0.1 mg/ml) before pelleting as before. 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 5 minutes on ice with vortexing to disrupt the insoluble pellet. The high salt extracted insoluble material was then pelleted at 4°C for 10 min at 5,000 g. The insoluble pellet was then washed with 0.5 ml DNase Digestion Buffer (40 mM Tris-HCl pH 7.9, 10 mM NaCl, 6 mM MgCl2, 1mM CaCl2, 300 mM sucrose, 0.05% NP-40, 0.5mM PMSF). Insoluble material was again suspended in 0.5 ml DNase Digestion Buffer before addition of 5 µl DNase I (Roche). Nuclei were incubated 30 minutes at 37°C before the addition of an equal volume 4 M High Salt Stop Buffer (10 mM Tis-HCl pH 7.5, 300 mM sucrose, 4M NaCl, 20 mM EDTA). The highly digested, insoluble chromatin was pelleted by centrifugation at 4°C for 10 min at 10,000 g. Time of digestion was titrated to yield majority subnucleosomal DNA fragments. The insoluble pellet was washed once more in in Isotonic Lysis Buffer lacking NP-40 and again harvested by centrifugation. RNA was removed from fragmented, insoluble genomic DNA by the addition of RNase A  (ThermoFisher, 0.1 mg/ml) in TE buffer and incubation for 30 minutes at 37°C. Remaining protein was then digested by the addition of SDS to 0.5 % and Proteinase K to 1 mg/ml and incubating for 90 minutes at 37°C. Samples were the subjected to phenol:chloroform:isoamyl alcohol extraction and ethanol precipitated. Libraries were prepared from isolated DNA using the Hyper Prep Kit from Kapa Biosystems using NEB TruSeq adapters and indexed primers for amplification.