GSM8604638: TSA-seq_HeLa_SA_pulldown_rep2; Homo sapiens; ChIP-Seq
HeLa cells were grown in 15 cm dish and performed SA or HS treatments, then fixed by quickly pouring away growth media, adding freshly made 0.5 mg ml-1 DSP (Sigma-Aldrich) in PBS, and incubating at RT for 30-45 min. Cells were permeabilized with 0.5% PBST at RT for 15 min. Free aldehyde groups were quenched with 20 mM glycine in PBS at RT for 10 min. Cells were then incubated with 1.5% H2O2 in PBS at RT for 1 h. Cells were blocked with 10% formamide/2×SSC at RT for 10 min and then incubated with Digoxigenin (DIG) labeled probes targeting SatⅢ RNA at 37°C overnight. Cells were incubated with HRP conjugated anti-Dig monoclonal antibody (Abcam, 1:2,500 dilution) in 0.1% PBST at 4°C for 10-12 h. Cells were TSA-labeled with a working solution of 50% sucrose/PBS containing 0.0015% H2O2 and 25 µM tyramide-biotin at RT for 30 min. For each staining, a small portion of attached cells were scraped off and loaded onto coverslips for immunostaining to visualize the TSA labeling. Cells were briefly air-dried on the coverslip and fixed. IF of tyramide-biotin and SatⅢ RNA were performed by Streptavidin-Alexa Fluor 568 (Thermo Fisher Scientific) and Anti-Digoxigenin/Digoxin (DIG) Antibody, DyLight-488 (Vector Lab). Coverslip was mounted in mounting medium and checked by fluorescence microscopy. Cells were scrapped and transferred for subsequent processing. Cells were incubated at 37°C for 30 min to reverse the DSP crosslinks. Cell lysis was done at 55°C with 500 μg ml-1 proteinase K for 6-12 h. DNA was extracted using an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) and with an equal volume of chloroform/isoamyl alcohol (24:1). Tubes were kept at 55°C for 1 h with the cap off to evaporate phenol residues, and then were treated with 25 μg ml-1 RNaseA at 37°C for 1 h and extracted again. DNA was then precipitated with 1:10 volume of 3 M Na acetate, pH 5.2, and two volumes of 100% ethanol at -20°C for 2 h. DNA was dissolved in double-distilled H2O and sonicated to 300-500 bp. DNA was then precipitated with 1:10 volume of 3 M Na acetate, pH 5.2, and two volumes of 100% ethanol at -20°C for 2 h. DNA was dissolved in double-distilled H2O, DNA concentration was measured by NanoDrop (Thermo Fisher Scientific). 150 μl streptavidin beads were washed in 1 mL W&B buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl) by rotating for 1 min. Beads were resuspended with DNA solution and rotated for 30 min at RT, then overnight at 4°C. Beads were washed with W&B buffer, TSE 500 buffer (20 mM Tris-HCl pH 8.0, 1% (v/v) Triton-X100, 0.1% (w/v) SDS, 2 mM EDTA, 500 mM NaCl), high TE buffer (10 mM Tris-HCl, 10 mM EDTA pH 8.0). Sequentially, each time removing the previous wash after immobilization of the beads on a magnetic stand. After the final wash, beads were resuspended in digestion mixture (10 mM Tris,10 mM EDTA, 0.5% SDS, 1 mg ml-1 proteinase K) overnight at 55°C with rotation. DNA was then extracted using ChIP DNA Clean & Concentrator kit (Qiagen). The quality of the DNA samples was checked using Agilent 2100 Bioanalyzer (Agilent), after which libraries were constructed according to the Illumina TruSeq ChIP Sample Preparation guide.