GSM9040776: Large HEK Torin; Homo sapiens; OTHER
For each experiment, 10 million cells were lysed in 1 mL RIPA buffer (50mM HEPES pH 7.4, 100mM NaCl, 1% NP-40, 0.5% Na-Deoxycholate, 0.1% SDS) supplemented with 20 μL Protease Inhibitor Cocktail (Sigma, #P8340-5mL), 10 μL of Turbo DNase (Invitrogen, #AM2238), 1X Manganese/Calcium mix (2.5 mM MnCl2, 0.5 mM CaCl2), and 5 μL of RiboLock RNase Inhibitor (Thermo Fisher, #EO0382)). Samples were incubated on ice for 10 minutes to allow lysis to proceed. After lysis, cells were sonicated at 3-4 W of power for 3 minutes (pulses 0.7 s on, 3.3 s off) using the Branson sonicator and then incubated at 37°C for 10 minutes to allow for DNase digestion. DNase reaction was quenched with addition of 0.25 M EDTA/EGTA mix for a final concentration of 10 mM EDTA/EGTA. RNase If (NEB, #M0243L) was then added at a 1:500 dilution and samples were incubated at 37°C for 10 minutes to allow partial fragmentation of RNA to obtain RNAs of approximately ~300-400 bp in length. RNase reaction was quenched with addition of 500 μL ice cold RIPA buffer supplemented with 20 μL Protease Inhibitor Cocktail and 5 μL of RiboLock RNase Inhibitor, followed by incubation on ice for 3 minutes. Lysates were then cleared by centrifugation at 15000 x g at 4°C for 2 minutes. The supernatant was transferred to new tubes and diluted in additional RIPA buffer such that the final volume corresponded to 1 mL lysate for every 100 μL of Protein G beads used. Lysate was then combined with the labeled antibody-bead pool and 1 M biotin was added to a final concentration of 10 mM as to quench any disassociated streptavidin-coupled oligos. Beads were left rotating overnight at 4°C on a HulaMixer. Following immunoprecipitation, beads were washed twice with RIPA buffer, twice with high salt wash buffer (50 mM HEPES pH 7.4, 1 M NaCl, 1% NP-40, 0.5% Na-Deoxycholate, 0.1% SDS), and twice with Tween buffer (50 mM HEPES pH 7.4, 0.1% Tween-20). After split-and-pool barcoding, beads were aliquoted into 5% aliquots for library preparation and sequencing. RNA in each aliquot was degraded by incubating with RNase H (NEB, #M0297L) and RNase cocktail (Invitrogen, #AM2286) at 37°C for 20 minutes. 3' ends of the resulting cDNA were ligated to attach dsDNA oligos containing library amplification sequences using a “splint” ligation as previously described (Quinodoz et al 2021)31. The “splint” ligation reaction was performed with 1X Instant Sticky End Master Mix (NEB #M0370) at 24°C for 1 hour with shaking at 1400 rpm on a ThermoMixer. Barcoded cDNA and biotinylated oligo tags were then eluted from beads by boiling in NLS elution buffer (20 mM Tris-HCl pH 7.5, 10 mM EDTA, 2% N-lauroylsarcosine, 2.5 mM TCEP) for 6 minutes at 91°C, with shaking at 1350 rpm. Biotinylated oligo tags were first captured by diluting the eluant in 1X oligo binding buffer (0.5X PBST, 5 mM Tris pH 8.0, 0.5 mM EDTA, 1M NaCl) and subsequently binding to MyOne Streptavidin C1 Dynabeads (Invitrogen, #65001) at room temperature for 30 minutes. Beads were placed on a magnet and the supernatant, containing cDNA, was moved to a separate tube. Biotinylated oligo tags were amplified on-bead using 2X Q5 Hot-Start Mastermix (NEB #M0494) with primers that add the indexed full Illumina adaptor sequences.