GSM8691715: TFEB_Torin1_Input biol rep 2; Homo sapiens; ChIP-Seq
After treatment, media on each 15 cm plate was changed to 20 mL of serumfree F-12K media containing 1% formaldehyde and incubated with shaking for 5 min. Cross-linking was then quenched by adding 10 mL of 0.125 M glycine in PBS and shaking for an additional 5 min. Media was then poured off, and plates were rinsed twice with ice-cold PBS. Finally, 5 mL of PBS plus protease inhibitors (0.25μM PMSF, 10 μg/mL aprotinin) was added to each plate, and cells were scraped on ice, collected in 15 mL conical tubes, and centrifuged at 1200 rpm for 10 min at 4°C. The supernatant was aspirated, and cell pellets were flash-frozen in liquid nitrogen and stored at −80°C until further processing. Plating, treatment, and fixation of cells were repeated twice for two biological replicates of each condition. Cell pellets were thawed on ice, resuspended in 2 mL of cell lysis buffer (5 mM PIPES at pH 8.0, 85 mM KCl, 0.5% NP-40; 1 mL/15 cm plate) with protease inhibitors and incubated for 10 min on ice. During incubation, the lysates were pipetted up and down every 5 min. Lysates were then centrifuged at 4000 rpm for 10 min. Nuclear pellets were resuspended in 6 vol of sonication buffer (50 mM Tris-HCl at pH 8.1, 10 mM EDTA, 0.1% SDS) with protease inhibitors, incubated on ice, and sonicated to obtain DNA fragments <1000 bp in length (Covaris S220 sonicator: 20% duty factor, 200 cycles/burst, 150 peak incident power, six to eight cycles of 30 sec on and 30 sec off). Sonicated lysates were cleared by centrifugation, and chromatin (400 μg/antibody) was diluted in RIPA buffer (10 mM Tris-HCl at pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-1000, 0.1% SDS, 0.1% Na-deoxycholate, 140 mM NaCl) to a final concentration of 0.8 mg/mL and precleared with magnetic Protein G Dynabeads (Thermo Fisher 10009D) for 2 h at 4°C. Precleared lysates were then immunoprecipitated overnight with 4 mg of normal antimouse IgG (Jackson ImmunoResearch 211-032-171) and anti-FLAG [M2] (Sigma Aldrich F1804). About 4% of the precleared chromatin was saved as input. Immunoprecipitated DNA was purified with Qiagen QIAquick PCR purification kit (Qiagen 28106) and eluted in 40 μL of 0.1× TE (1 mM Tris-HCl at pH 8.0, 0.01 mM EDTA). Samples were checked by qPCR together with 2% of the input chromatin and then used in ChIP-seq library preparation. ChIP-seq libraries were prepared independently from two ChIP biological replicates using the NEBNext Ultra II DNA library preparation kit (New England Biolabs E6177L) according to the manufacturer's instructions with a few modifications. As starting material, 20 ng of ChIP input DNA (as measured by NanoDrop) and 20 μL of the immunoprecipitated DNA (spiked with 5 μL of 10 ng/mL sheared Drosophila melanogaster DNA) was used. The recommended reagents' volumes were cut in half. The NEBNext Adaptor for Illumina was diluted 1:10 in Tris/NaCl (pH 8.0; 10 mM Tris-HCl at pH 8.0, 10 mM NaCl), and the ligation step was extended to 30min. A single purification step with 0.9× vol of Agencourt AMPure XP PCR purification beads (Beckman Coulter A63880) was performed after ligation. DNA was eluted in 22 μL of 10 mM Tris-HCl (pH 8.0), and 20 μL of the eluted DNA was used for the library enrichment step. Library samples were enriched with 11 cycles of PCR amplification in 50 μL of total reaction volume (10 μL of 5× KAPA buffer, 1.5 μL of 10 mM dNTPs, 0.5 μL of 10 mM NEB Universal PCR primer, 0.5 μL of 10 mM index primers, 1 mL of KAPA polymerase, 16.5 μL of nuclease-free water, 20 μL of sample). After amplification, PCR samples were again purified with 0.9× vol of AMPure XP PCR amplification beads and eluted in 33 μL of 10 mM Tris-HCl (pH 8.0). Library concentration was assessed using Qubit dsDNA HS assay kit (Invitrogen Q32851)