GSM8162570: hek293-a; Homo sapiens; OTHER
HEK293 and HeLa cells (human cervical cancer) were purchased from the American Type Culture Collection (ATCC) and confirmed via short tandem repeat (STR) profiling with CellBank Australia. The cells were grown in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1× antibiotic-antimycotic solution (Sigma). Cells were cultured following the protocol previously described​ (Sneddon et al. 2022). Namely, cell cultures were propagated in a 1:3 split, with replenishment of media every 4 days. Cell culture, cell pellet collection, cell lysis, polyadenylated RNA extraction and RNA cleanup with SPRI beads were performed as previously described (Sneddon et al. 2022)​.   The immortalized human peripheral vein-derived B-cells GM12878 and B-cell acute lymphocytic leukemia (B-ALL)-derived REH and KOPN8 cell lines were obtained from the Coriell Institute and cultured with 10% FBS-supplemented RPMI1640 media (Gibco) in T25 flasks (Corning) under 5% CO2 and 37°C. Cells were harvested by a 5 minute centrifugation at 300rcf at 4°C. The resultant cell pellet was washed with ice-cold PBS and separated by another centrifugation at 300rcf for 5 minutes and aspirated. The protocols for subculturing, cell lysis and total RNA extraction were performed as described for HEK293 and HeLa cell lines.   The lymphoblastoid cell line (LCL) GM24385 (Corielle Institute) was grown in RPMI1640 media (Gibco) supplemented with 15% Hi-FCS and 2 mM L-Glutamine in 6-well plates (Coning) under 5% CO2. Cells were harvested at a density of 106 cells/ml. Cell pellet collection was performed by transferring GM24385 cell suspension into 15 ml conical centrifuge tubes (Falcon) and centrifuging at 500×g for 10 minutes at room temperature.   To isolate RNA from the cytoplasmic and nuclei fractions, 107 cells were lysed in 200 µl of the non-denaturing lysis buffer containing 25 mM HEPES-KOH (pH 7.6 at 25°C), 50 mM KCl, 5.1 mM MgCl2, 2 mM DTT, 0.1 mM EDTA, 5% v/v glycerol, 2× Complete EDTA-free protease inhibitor and 0.5% v/v Igepal CA-630B. Cells were resuspended in the lysis buffer through pipetting and RNasin Plus (Promega) was immediately added to the final concentration of 1 U/µl. Cell lysis was completed by passing the lysate 4× through a 20-gauge needle followed by passing it 4× through a 27-gauge needle. The cell lysate was then centrifuged at 1,000× g for 5 minutes at 4°C. The supernatant was transferred into a new 1.5ml tube and mixed with 350µl of the RA1 lysis buffer, followed by RNA isolation using silica columns (Macherey Nagel) to obtain the cytoplasmic RNA fraction. The process included on-column Dnase treatment with TURBO Dnase (Thermo Fisher Scientific). The pelleted nuclei were resuspended in 1 ml of ice-cold sterile PBS and 50 µl counted using cell counter (Beckman-Coulter). The nuclei suspension was spun again at 1,000× g for 5 minutes at 4°C, the supernatant aspirated and 6.6×106 nuclei were lysed in 700 µl of the RA1 RNA lysis buffer and isolated using the silica columns as described above. RNA isolated from the cytoplasmic fraction was eluted from the columns in 80µl, and from nuclei in 120µl of Rnase-free water, and stored at -80°C.   For in vitro polyadenylation, ~9µg of the RNA in 94 µl of deionized water or 25mM HEPES-KOH (pH 7.6 at 25°C), 0.1 mM EDTA (HE) buffer were first denatured by incubating at 65°C for 3 minutes and immediately chilling in ice. The solution was then supplemented with 12µl of 10× E. coli Poly(A) Polymerase buffer (New England Biolabs), 8µl of 1mM ATP and mixed. To the resultant solution, 3µl of 40U/µl Rnasin Plus (Promega) and 3µl of 5U/µl E. coli Poly(A) Polymerase (New England Biolabs) were added and mixed, and the resultant mixture incubated at 37°C for 30 minutes. The eluate from in vitro polyadenylated RNA was further purified following the protocol previously described (Sneddon et al. 2022)​ for RNA cleanup with SPRI beads. For all samples, the flow cell priming and library sequencing protocol were performed as previously described​ (Sneddon et al. 2022)​. Nanopore sequencing was performed using an ONT MinION Mk1B with R9.4.1 flow cell for 24 hours. The default settings for the MinKNOW software (standalone GUI v5.7.10, core v5.7.2) were used and the SQK-RNA002 kit was selected.   Additionally, for the sample hek293_liverun RISER was run at the same time as the MinKNOW sequencing run. In the RISER code, the flow cell channels were split into two groups to remove the effect of inter-flow cell variability by simultaneously testing two conditions: (1) RISER depleting mRNA and mtRNA, and (2) no RISER (control). To split the flow cell channels, the channel numbers divisible by 2 were used for the RISER deplete condition (1) while the remaining channels were used for the control condition (2). RISER was started after the first pore scan finished and was run for the same duration as the sequencing run; 24 hours. The live run was performed on a computer running Ubuntu 20.04 with one NVIDIA GeForce GTX 1650 Ti GPU and python v3.8.10. Nanopore direct RNA sequencing