SAFB associates with nascent RNAs and can promote gene expression in mouse embryonic stem cells [RIP-seq]

RNA-IPs in WT and SAFB1/2 DKO mESCs. RIPs were performed essentially as in (Schertzer et al. 2019a), which is a protocol originally adapted from (Hendrickson et al. 2016; Raab et al. 2019). 25μL protein A/G agarose beads (Santa Cruz sc-2003) were washed three times in blocking buffer (0.5% BSA in 1xPBS) and incubated overnight at 4°C with 10μL antibody (anti-SAFB; Bethyl 812-300A; FLAG, Sigma F1804; V5, Sigma V8012) or 10μg of mouse IgG (Invitrogen). 10x10^6 cells were resuspended in 500μL RIPA Buffer (50mM Tris-HCl, pH8, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 5mM EDTA, 150mM KCl) supplemented with 1x Protease Inhibitor Cocktail (PIC; Sigma Product #P8340), 2.5μL SuperaseIN (Thermo Fisher Scientific AM2696) and 0.5mM DTT and sonicated twice 30s on and 1 min off at 30% output using the Sonics Vibracell Sonicator (Model VCX130, Serial# 52223R). Samples were spun down at high speed and 50μL total lysate was saved for input. Beads were washed three times in 1mL fRIP buffer (25mM Tris-HCl pH 7.5, 5mM EDTA, 0.5% NP-40, 150mM KCl) and resuspended in 450μl fRIP buffer supplemented as above with PIC, SuperaseIN and DTT, then mixed with sonicated samples. Samples were rotated overnight at 4°C, then washed once with 1mL fRIP buffer and resuspended in 1mL PolII ChIP Buffer (50mM Tris-HCl pH 7.5, 140mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X-100, 0.1% Sodium-deoxycholate, 0.1% Sodium dodecyl sulfide) before transferring to a new 1.5mL tube. Samples were rotated at 4°C for five minutes, spun down at 1200xg, and the supernatant aspirated. Samples were washed twice more with 1mL PolII ChIP Buffer, twice with 1mL High Salt ChIP Buffer (50mM Tris-HCl pH 7.5, 500mM NaCl, 1mM EDTA, 1mM EGTA, 0.1% sodium-deoxycholate, 0.1% sodium dodecyl sulfide, 1% Triton X-100), and once in 1mL LiCl buffer (20mM Tris pH 8.0, 1mM EDTA, 250mM LiCl, 0.5% NP-40, 0.5% sodium-deoxycholate); each wash included a five-minute rotation at 4°C. At the final wash, samples were transferred to a new 1.5mL tube. After the final wash, inputs were thawed and bead samples were resuspended in 56μL water, 33μL of 3x reverse-crosslinking buffer [3x PBS, 6% N-lauroyl sarcosine and 30mM EDTA], 5μL 100mM DTT, 20μL Proteinase K, and 1μL of SUPERaseIN. Samples were incubated for 1hr at 42°C, then 1hr at 55°C, then 65°C for 30 minutes, and mixed by pipetting every 15 minutes. Afterwards, 1mL Trizol was added, samples were vortexed, 200μL CHCl3 was added, samples were vortexed, and finally spun at 12000xg for 15 mins at 4°C. The aqueous phase was then extracted and one volume of 100% ethanol was added. Samples were vortexed and applied to Zymo-Spin IC Columns (from #R1013) and spun for 30 seconds at top speed on a benchtop microcentrifuge. 400μL of RNA Wash Buffer (Zymo #R1013) was added and samples were spun at top speed for 30 seconds. For each sample, 5μL DNAse I and 35μL of DNA Digestion Buffer (Zymo #R1013) was added directly to the column matrix and incubated at room temp for 20 minutes. 400μL of RNA Prep Buffer was then added (Zymo #R1013), and columns were spun at top speed for 30 seconds. 700μL RNA Wash Buffer (Zymo #R1013) was then added, and columns were spun at top speed for 30 seconds. 400μL RNA Wash Buffer was then added, and columns were spun at top speed for 30 seconds. The buffer reservoir was then emptied and columns spun again for 2 minutes to remove all traces of wash buffer. Column were transferred to a clean tube, 15μL of ddH2O was added to each, and after a five-minute incubation, samples were spun at top speed to elute. For RIP-Seq inputs, 100ng of RNA prepared from RIP input samples was used for library preparations. For RIP-Seq RIP samples, 9μL of RIP sample (from 15μL total) were used. Each library preparation included 1μL of 1:250 dilution of ERCC Spike-In RNAs (Ambion #4456653). 10μL total were prepped using the KAPA RNA HyperPrep Kit with RiboErase (Kapa Biosystems; product #KR1351). Sequencing was performed on an Illumina Next-Seq 500, using high-output, 75-cycle kits.