title: GSM2563800: nasBS-seq_chase_daughter_rep3; Homo sapiens; Bisulfite-Seq
library_construction_protocol: For pulse, H9 cells were transferred to medium containing 50 µM EdU and were cultured at 37 °C for 20 min before formaldehyde crosslinking. For chase, after labeling with EdU, cells were washed with DPBS twice, and cultured in medium containing 50 µM dT for 8 hr before formaldehyde crosslinking. Crosslinking with 1% formaldehyde was done in DPBS at RT for 10 min and was quenched with 0.125 M glycine. Cells were collected and washed with DPBS, and lysed in Lysis Buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, 10% glycerol). Pellets were collected and re-suspended in Sonication Buffer (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 0.1% sodium deoxycholate, 0.5% sodium lauroylsarcosine). Sonication was done on ice using Bioruptor. Triton X-100 was added to a final concentration of 1% before centrifugation at 15,000 xg. Supernatant was collected as fraction of chromatin. Elution Buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1% SDS) was added to chromatin in a 1:1 ratio. Crosslinking reversal was done by incubation at 65 °C for 8 hr. Proteinase K was added to a final concentration of 0.2 mg/ml before incubation at 55 °C for 30 min. Phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation was done sequentially to purify genomic DNA. Click chemistry was performed in a 200 µl volume (174 µl DNA in DPBS, 2 µl 1 mM biotin-azide, 4 µl 100 mM CuSO4, 20 µl 100 mM sodium ascorbate) at 37 °C for 1 hr. After ethanol precipitation, DNA was incubated with Dynabeads Streptavidin C1 beads on a rotor for 30 min at RT, and washed according to the manufacturer’s instructions. Beads were incubated with End Repair Enzyme Mix on a rotor for 30 min at RT. After washing, beads were incubated with Klenow Fragment (3'→5' exo-) reaction mix on a rotor for 30 min at RT. After washing, beads were incubated with T4 DNA ligase reaction mix and methylated adaptors on a rotor for 2 hr at RT. After washing, beads were incubated in 150 mM NaOH for 3 min at RT. Supernatant was collected as the fraction containing the parental strand DNA (bio-). Beads were washed 3 times with NaOH, re-suspended in 95% formamide and 10 mM EDTA pH 8.2, and incubated at 90 °C for 3 min. Supernatant was collected as the fraction containing the daughter strand DNA (bio+). Both fractions were ethanol precipitated, mixed with 1 pg of lambda DNA, and bisulfite converted with the EZ DNA Methylation-Lightning Kit. PCR amplification was done with HiFi Uracil+ polymerase. For each library, the minimal number of PCR cycles were used to yield at least 20 ng of DNA measured after purification with AMpure XP beads.
attributes.tag: GEO Accession
attributes.value: GSM2563800