GSM4455860: TLA_GFP_u4; Homo sapiens; OTHER
Samples of 10 million cells were crosslinked in 2% formaldehyde for 10 minutes at room temperature and quenched with glycine to a final concentration of 275 mM. Cell were lysed for 5 minutes at room temperature in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, 1x protease inhibitors). Crosslinked samples were digested at 37ºC overnight with 400 U NlaIII (NEB) followed by NlaIII heat inactivation. Samples were ligated at room temperature for 2 hours with 20 U T4 DNA ligase (Thermo Scientific) in 500 µL. Ligated samples were de-crosslinked with 5 µL Proteinase K (10 mg/mL) at 65ºC overnight followed by RNAse A treatment and phenol-chloroform purification. Samples then digested overnight at 37ºC with 50 U NspI (NEB) followed by NspI inactivation. The second ligation was performed overnight at 16ºC with 100 U T4 DNA ligase in 14 mL. The resulting TLA circularized templates were purified with the QIAquick PCR purification kit. TLA libraries were generated by amplifying 800 ng of purified TLA template with TLA viewpoint primers. 2 x 150 bp paired-end sequencing of pooled TLA libraries was performed on Illumina HiSeq 4000.
GEO Accession
GSM4455860