title: GSM5494640: HeLa-S3 INPUT rep2; Homo sapiens; OTHER
library_construction_protocol: Hela cells from one R150 dish was collected by trypsinization and lysed by 4ml DNAzol with the addition of DTT to a final concentration of 10 mM. Genomic DNA were precipitated following the manufacturer's instruction. DNA pellets were resolved in 300 μl of Alkaline Resolving Buffer (8 mM NaOH and 100 μM TEMPO). Typically, more than 200 μg of genomic DNA were obtained from one dish, which were used for ELISA or/and snOG-seq immediately. Notably, all buffers and reagents used in DNA extraction, ELISA and chemical labeling steps were prepared with supelco-grade deionized water in order to prevent further DNA oxidation during these steps, while DTT and TEMPO were added for the same purpose. Selective chemical labeling: Extracted DNA (8-10 μg) were incubated with 20 mM of NH2-(PEG)11-biotin and 5 mM of K2IrBr6 in the presence of sodium phosphate (100 mM, pH 8.0) for 5 min on ice. The reaction was terminated by passing through a Microspin G50 column (GE) which was pre-washed with ice-cold deionized water, then labeled DNA were ethanol precipitated and dissolved in 1xTE buffer. Because artificial DNA oxidation after labeling reaction couldn't interfere with following steps, biotinylated genomic DNA could be stored at -20°C until next step; Fragmentation, End-repair and first adaptor ligation: These steps were performed as previously described. Briefly, biotinylated DNA were sheared by a Qsonica Q800 Sonicator to generate fragments averaging 600 bp in length. Purified DNA fragments (up to 5 µg) were used for End-repair and dA-tailing by NEBNext DNA Library Prep Kit following the manufacture's instruction. Subsequently, Ad1 (100 pmol/µg of input DNA) was ligated to both ends by NEBNext T4 Quick Ligase at 4°C overnight. The ligation products were purified by 0.7 × NEBselect beads and eluted in 15 µl 0.1x TE; Affinity purification and primer extension  Each samples was incubated with 5 µl of Dynabeads MyOne Streptavidin C1 beads in 1x binding buffer in the presence of 10 µg of salmon sperm DNA at 4°C for 1 h. Then the beads were sequentially washed twice with 1x B&W buffer, twice with OG-wash buffer IV (100 mM pH 8.0 Tris-Cl, 1 mM EDTA, 0.5 M LiCl, 1% CA630, 1% sodium deoxycholate), twice with wash buffer V (1 mM pH 8.0 Tris-Cl, 0.1 mM EDTA, 100 mM NaCl, 2% SDS) , one time with 1X TE and finally one time with 0.1X TE, followed by resuspending in 6 µl 0.1x TE. For primer extension reaction, 30 pmol of Extension Primer was attached to biotinylated DNA which were still bound on beads and extended by KAPA HiFi DNA polymerase (98°C 20 s, 62°C 15 s, 72°C 15 s). Excess primers were degraded by incubating with 1.5 µL of ExoI at 37°C for 10 min. The reaction was ended by adding equal volume of 1% SDS. Next, the beads were sequentially washed twice with 1x B&W buffer, twice with OG-wash buffer IV and twice with wash buffer V. Extension products were released from templates by incubation with fresh NaOH elution buffer (0.1 M NaOH plus 0.1% CA630) at room temperature for 2 min to release the complementary strands. The collected DNA were neutralized with 0.5M HAc and purified by ethanol precipitation; Second adaptor ligation, PCR amplification and high-throughput sequencing: The precipitated DNA were dissolved and subjected to ligation with 40 pmol of Ad2 by Instant Stick Ends Ligase Master Mix (NEB) at 4°C overnight. The ligation products were purified by 0.8 × NEB select beads. Two percent of ligated DNA were used for library quality check while the rest were amplified with NEBNext index primer set for Illumina by NEBNext UltraII Q5 DNA polymerase following the manufacturer's instruction to generate sequencing libraries. Qualified libraries were pooled and sequenced on Illumina Hiseq X platform by WuXiNextCODE Company.
attributes.tag: GEO Accession
attributes.value: GSM5494640