GSM5399032: siGL3 cytoDRIP-seq -S9.6 replicate 1; Homo sapiens; OTHER
Cells were harvested using trypsin, washed in PBS and pelleted by centrifugation. Cells were fractionated using the Nuclear and Cytoplasmic Extraction kit (Thermo Fisher Scientific) according to manufacturer's instructions. Cytoplasmic fractions were recovered, and small amounts taken for western blotting and protein quantification, and the rest incubated in 0.4% SDS and 40 μg/ml of proteinase K (Thermo Fisher Scientific) for 90 mins at 37°C, followed by phenol-chloroform extraction and ethanol precipitation. Samples were resuspended in ultrapure distilled water, adjusted to 550 mM NaCl and treated with RNase A (1μg/mL) for 25 min, followed by phenol-chloroform extraction and ethanol precipitation. For immunoprecipitation, 16 μg of S9.6 antibody or mouse IgG were bound to Dynabeads Protein G beads (Thermo Fisher Scientific) in 1× binding buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 1% Triton X-100, 150 mM NaCl, 0.5% sodium deoxycholate) for 4-6h at 4°C. In parallel, samples were resuspended in 1X TE buffer and then pre-cleared with Dynabeads Protein G for 1-2 h in 1× binding buffer. Pre-cleared genomic samples were then added to S9.6 or IgG-bound beads and incubated overnight with rotation at 4°C. Bound beads were washed thrice with TE and then thrice with TSE buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 150 mM NaCl). Elution was performed in 200μL elution buffer (50 mM Tris pH 8, 10 mM EDTA, 0.5% SDS, 8 μl Proteinase K 20 mg/ml) for 50 min at 50°C. After elution, genomic material was resuspended in 130 μl TE buffer and sonicated in 6 × 16 mm microtubes (Covaris, Woburn, MA, USA) to a peak fragment size of 300 bp, performed on a Covaris machine (E220 evolution) using SonoLab 7.3 software) (10% Duty Factor, 200 cycles/burst, 140 peak incident power, 30s per tube). DNA libraries were synthesized from ssDNA using the Accel-NGS 1S DNA library kit (Swift Biosciences, Ann Arbor, MI, USA), according to the manufacturer's protocol. Using multiplexing adapters from the 1S Plus Set A Indexing Kit (Swift Biosciences), adapter-ligated DNA was amplified by PCR. Following PCR, DNA fragments 200–600 bp were retained by size selection using a 0.6× volume ratio of AMPure XP beads (Beckman Coulter)/sample followed by 1.0× ratio. Library DNA was analyzed on a Bioanalyzer DNA HS (Agilent, Santa Clara, CA, USA) at the Stanford Protein and Nucleic Acid Facility, quantified by qPCR using NEBNext Library Quant Kit for Illumina (New England Biolabs)