GSM8816543: HEK_293_rep2; Homo sapiens; OTHER
sBLISS was conducted as previously described33,60. In summary, 106 cells were fixed in 2% paraformaldehyde in 10% FCS/PBS for 10 minutes at room temperature. The fixation was quenched with 125 mM glycine for 5 minutes at room temperature, followed by another 5 minutes on ice and two washes in ice-cold PBS. Cells were lysed for 60 minutes on ice, and their nuclei were permeabilized for 60 minutes at 37°C. Next, nuclei were rinsed twice with CutSmart Buffer containing 0.1% Triton X-100 (CS/TX100), and double-strand break (DSB) ends were blunted in situ using NEB's Quick Blunting Kit for 60 minutes at room temperature. The blunted nuclei were then washed twice with 1x CS/TX100 before in situ ligation of the sBLISS adapters to the DSB ends. Adaptor ligation was carried out with T4 DNA Ligase for 20-24 hours at 16°C, with BSA and ATP added. Following ligation, the nuclei underwent two washes with 1x CS/TX100, and genomic DNA was extracted using Proteinase K at 55°C for 14-18 hours while shaking at 800 rpm. Proteinase K was then heat-inactivated for 10 minutes at 95°C, followed by extraction using Phenol:Chloroform:Isoamyl Alcohol, Chloroform, and ethanol precipitation. The purified DNA was sonicated in 100 μL of ultra-pure water using Covaris M220 for 60 seconds. Sonicated samples were concentrated with AMPure XP beads (Beckman Coulter), and fragment sizes were evaluated using a BioAnalyzer 2100 (Agilent Technologies), targeting a range of 300 bp to 800 bp with a peak around 400-600 bp. The sonicated DNA was then in vitro transcribed using the MEGAscript T7 Kit for 14 hours at 37°C. After RNA purification and ligation of the 3'-Illumina adaptors, the RNA underwent reverse transcription. The final library indexing and amplification step was performed with NEBNext® Ultra™ II Q5® Master Mix.MCF7 cells (~106) were crosslinked with 1% formaldehyde (methanol free, Thermo Scientific 28906) for 10 min at room temperature and quenched with glycine, 125 mM final concentration. Fixed cells were washed twice in PBS and incubated in sonication buffer (0.5M NaCl, 0.5%SDS RIPA buffer containing PMSF, protease and phosphatase inhibitors) for 30min on ice. Cells were sonicated using Covaris M220 for 10min to produce chromatin fragments of ~200-300 bp. The sheared chromatin was centrifuged 10min at maximum speed. From the supernatant, 50ul were saved as input DNA and the rest was diluted in RIPA buffer without NaCl and SDS. The chromatin was immunoprecipitated by incubation with 5 μg of gamma-H2AX (phospho S139; Abcam, AB2893) antibody pre mixed with 100ul Dynabeads Protein G (ThermoFisher, 10004D) and incubated over-night at 4°C with rotation. Immunoprecipitates were washed twice with RIPA buffer + 150mM NaCl, twice with RIPA buffer + 300 mM NaCl, and twice with TE buffer. The chromatin was eluted from the beads with 200ul of direct elution buffer (10mM Tris pH 8, 0.3M NaCl, 5mM EDTA, 0.5% SDS) and incubated overnight at 65°C to reverse the cross-linking. Samples were treated with RNAse for 1h at 37 °C and with proteinase K at 55 °C for 2 h. DNA was cleaned up by QIAquick PCR purification column (Qiagen), according to the manufacturer's instructions, and eluted in 30 μl of elution buffer. The ChIPed and the Input DNA were used for real-time PCR or to prepare libraries by Truseq (Ilumina) and sequenced in Nextseq (Illumina). sBLISS fastq files are initially de-multiplexed using sample barcodes. Quality control is performed with trim galore to eliminate residual adapters, trim reads to a base quality of at least 20, and filter out short reads smaller than 20 bp. Initial and final sample qualities are assessed with fastqc. Quality-processed fastq files are aligned to the GRCh38 assembly with hisat2, then sorted and indexed using samtools. The resulting bam files are de-duplicated with umi-tools, utilizing genomic coordinates and Unique Molecular Identifiers (UMI). Custom Python and R scripts are employed to identify read start positions and convert bam files to bigwig format for downstream analysis. An additional custom R script is used to discard a blacklist of positions, primarily within centromeres.