title: GSM8792074: Lamin A/C knockout Hela cells, BrdU-seq, biol rep 2; Homo sapiens; OTHER
library_name: GSM8792074
library_construction_protocol: Hela cells were cultured in 10 cm dishes at a density of ~20%, then synchronized at either the G1/S boundary or specific points in S phase. After synchronization, cells were washed three times with PBS. BrdU (Sigma-Aldrich, B5002) was added to the medium to label replicating DNA for specific durations. Following labeling, cells were washed with PBS, and lysed in RIPA buffer (Yeasen, 20101ES60) containing RNase A (Qiagen, 19101) and Proteinase K (Thermo Fisher Scientific, EO0491) at final concentrations of 0.1 mg/mL and 0.2 mg/mL, respectively. Lysis was carried out in a thermomixer first at 37°C for 2 hours, followed by 55°C for 5 hours to ensure complete cell lysis and DNA release. Genomic DNA was extracted from the lysate using phenol-chloroform extraction to ensure purity. DNA was mixed with phenol-chloroform-isoamyl alcohol (24:25:1) at a 1:1 ratio, shaken for 5 minutes, and allowed to settle for phase separation. After centrifugation (15,000 g, 10 minutes), the upper aqueous phase containing the DNA was collected. The extraction was repeated, and the DNA was precipitated with ethanol and 3 M sodium acetate (pH 5.5). DNA was pelleted by centrifugation (16,000 g, 30 minutes at 4°C), washed with 75% ethanol, air-dried, and resuspended in ultrapure water. The extracted DNA was fragmented into ~200 bp fragments using a Covaris sonicator (175 W, 10% duty cycle, 200 cycles per burst). Fragmented DNA was then purified using Vazyme magnetic beads (Vazyme, N401-01). Following DNA fragmentation, BrdU-labeled DNA was denatured to single-stranded DNA by heat treatment. The denatured DNA was incubated with BrdU-specific antibodies (BD, 555627) in an immunoprecipitation buffer. Protein G-Dynabeads (Invitrogen, 10004D) were used to capture the antibody-DNA complexes. The immunoprecipitated DNA was washed extensively, and DNA was eluted with TE buffer. The recovered DNA fragments were used to generate sequencing libraries with the Vazyme single-strand DNA library preparation kit (Vazyme, NE103). Libraries were sequenced on an Illumina platform.