GSM8513141: OSRC2, PT2399, Rep1; Homo sapiens; OTHER
Cells (Human or Drosophila) were washed with RT PBS, trypsinized for 1-2 min at 37°C, quenched with ice-cold DMEM +10% FBS and transferred to 50 ml conical tubes. After harvesting, cells were always kept on ice. 5×106 to 1×107 cells per condition were collected by centrifugation at 300×g for 4 min at 4°C. The supernatant was removed, and the cell pellets were gently resuspended in 10 ml ice-cold PBS, followed by centrifugation at 300×g for 4 min at 4°C. The supernatant was removed, and the cells were resuspended in 250 ml wash buffer [10 mM Tris-HCl pH 8.0, 10 mM KCl, 250 mM Sucrose, 5 mM MgCl2, 1 mM EGTA, 10% (v/v) glycerol, 0.5 mM DTT (Thermo Fisher Scientific 707265ML), 0.2 ml/ml RNase inhibitor (Invitrogen AM2696) and 1× protease inhibitor (Thermo Fisher Scientific A32965)]. The cell pellets were gently pipetted using wide bore filtered pipette tips (Thermo Fisher Scientific 2079GPK) to create a single-cell suspensions. The cell suspension was passed through a cell strainer once (Falcon 352235) to avoid cell clumps. 10 ml permeabilization buffer [10 mM Tris-HCl pH 8.0, 10 mM KCl, 250 mM Sucrose, 5 mM MgCl2, 1 mM EGTA, 10% (v/v) glycerol, 0.1 % (v/v) IGEPAL CA-630, 0.05% (v/v) Tween-20, 0.5 mM DTT, 0.2 l/ml RNase inhibitor and 1× protease inhibitor] was slowly (over 5-10 seconds) added to the single-cell suspension along the wall of the conical tube. The samples were then incubated on ice for 5 min before centrifugation at 400×g for 4 min at 4°C. The supernatant was removed by gently inverting the tubes after which 1 ml of ice-cold wash buffer was added to each of the pellets to dilute out the residual permeabilization buffer followed by gently pipetting 2-3 times to achieve a single cell suspension. Next an additional 9 ml of ice-cold wash buffer was added to each tube and the samples were pelleted by centrifugation at 400×g for 4 min at 4°C. This wash procedure (1 ml wash buffer followed by 9 ml wash buffer) was repeated an additional 2 times. After the washes, the cell pellets were resuspended in 250 l freezing buffer [50 mM Tris-HCl, pH 8.0, 40% (v/v) glycerol, 5 mM MgCl2, 1.1 mM EDTA, 0.5 mM DTT, 1 l/ml RNase inhibitor] and transferred to 1.5 ml low binding tubes (Thermo Fisher Scientific Pl90410). 250 l freezing buffer/tube was also added to the 50 ml conical tubes, followed by gentle pipetting, to collect any residual cells, which were then pooled with the first resuspension (bringing the total volume to 500 l, which was further resuspended by gentle pipetting). Trypan blue-stained cell number was determined using a hemocytomer after which the cells were diluted using freezing buffer to the concentration of 1×106 cell/100 l. Aliquots of 5×106 permeabilized cells per 1.5 ml low binding tube were flash frozen in liquid nitrogen and stored at -80°C prior to use. To conduct nuclear run-ons and library preparation, aliquots of frozen permeabilized cells were thawed on ice, pipetted gently, and recounted using a Luna II automated cell counter (Logos Biosystems). For each sample, 1×106 permeabilized Human cells were used for nuclear run-ons, with 5×104 permeabilized Drosophila S2 cells added to each sample for normalization. Nuclear run-on assays and library preparation were performed as described in (Reimer et al, Mol Cell, 2021) with modifications. Briefly, nascent RNA was labeled by a run-on assay in which an equal volume of 2× nuclear run-on buffer [10 mM Tris-HCl, pH 8.0, 300 mM KCl, 1% sarkosyl, 10 mM MgCl2, 1 mM DTT, 20 M/ea biotin-11-NTPs (Perkin Elmer NEL543001EA), 0.8 U/l RNase inhibitor)] was added into permeabilized cell mixture and incubated for 5 min at 37°C. RNA was purified by the Total RNA Purification Kit (Norgen Biotek Corp 17200) per manufacturer's instructions, eluted in 100 l H2O, and fragmented by base hydrolysis by adding 25 l 5× fragmentation solution (375 mM Tris-HCl, pH 8.3, 562.5 mM KCl, 22.5 mM MgCl2) followed by incubation for 5 min at 94°C. Fragmentation was stopped by adding 125 l ice-cold 0.1 M EDTA. To enrich for biotin-labeled nascent RNA, 10 ml of streptavidin C1 magnetic beads (Invitrogen 65001) were washed, rendered RNase free per manufacturer's instructions, and resuspended in 50 ml binding buffer (10 mM Tris-HCl pH 7.4, 300 mM NaCl, 0.1% Triton X-100) before being added to the fragmented RNA. The samples were rotated end-to-end for 20 min at RT. The streptavidin magnetic beads were then washed twice with high salt buffer (50 mM Tris-HCl pH 7.4, 2 M NaCl, 0.5% Triton X-100), twice with binding buffer (listed above), and twice with low salt buffer (5 mM Tris-HCl pH 7.4, 0.1% Triton X-100). The beads were next resuspended in 500 ml TRIzol Reagent (Invitrogen 15596026) and incubated at 65°C for 5 min to elute RNA from the beads. This elution step was repeated once and the first and second eluates for a given sample were pooled. The RNA was subsequently purified by adding 2.75× volumes of 100% ethanol to the 400 l solution recovered from aqueous phase of TRIzol in the presence of 20 g of Ultrapure glycogen (Invitrogen 10814010). The pelleted RNA was subsequently washed per manufacturer's instructions and resuspended in 5 l of 10 M App-VRA3_6N 3' end adapter prepared using the 5' DNA Adenylation Kit (New England Biolabs M2611A) and ligated using T4 RNA ligase 2, truncated KQ (New England Biolabs M0373) per manufacturer's instructions with 15% (w/v) PEG-8000 in final solution and incubated overnight at 16°C. 180 ml of betaine binding buffer [1.42g of betaine (Sigma-Aldrich 61962) in 10 ml with binding buffer] was mixed with ligations and incubated for 5 min at 65°C and then for 2 min on ice. Capture on Streptavidin beads was again performed as above to enrich for ligated nascent RNAs except for the addition of a final wash with 1× MDE buffer (New England Biolabs B0608S) after the washes with high salt buffer, binding buffer, and low salt buffer. To prepare nascent RNA for 5' end adapter ligation, the 5' ends of the RNA were first decapped by mRNA decapping enzyme (New England Biolabs M0608S) for 1 h at 37°C. The beads were washed once in high, once with low salt buffer, and then once in 1× T4 PNK reaction buffer. The samples were then treated with T4 polynucleotide kinase (New England Biolabs M0201) for 1 h at 37°C for 5'-hydroxyl repairs, followed by one wash with high salt buffer, one wash with low salt buffer, and one wash with 0.25× T4 RNA ligase reaction buffer prior to resuspension in 5 ml of 10 pmol 5' RNA adapter VRA5_6N. Next T4 RNA ligase 1 (New England Biolabs M0204L) was used to ligate the 5' RNA adapter per manufacturer's instructions with 15% (w/v) PEG-8000 in final solution and incubated overnight at 16°C. Following the 5' end ligation, beads were washed twice with high salt buffer, twice with binding buffer, and twice with low salt buffer, then once with 0.25× SuperScript IV reaction buffer. Reverse transcription was performed using SuperScript IV Reverse Transcriptase (Invitrogen 18090010) and the cDNA was eluted from the beads by heating the samples twice for 30 seconds at 95°C. The eluted cDNA was initially amplified for 5-cycles of "preCR" [NEBNext Ultra II Q5 master mix (New England Biolabs M0544S) with Illumina TruSeq PCR primers RP-1 and RPI-X for indexing] following the manufacturer's suggested PCR cycling protocol for library construction]. A portion of preCR was serially diluted and for test amplification to determine optimal amplification of final libraries. Libraries were amplified by 3 additional cycles of PCR (8 cycles in total). The amplified library was purified using the ProNex Size-Selective Purification System (Promega) and sequenced using the Illumina NovaSeq platform.