title: GSM6500717: HeLa_M11B_WT; Homo sapiens; OTHER
library_construction_protocol: 3 WT and 3 NSUN2-KO 80% confluent 10 cm plates were washed twice in ice-cold PBS and scraped in 500 µl of denaturing lysis and binding buffer (100 mM Tris-HCl pH 7.4, 1 % w/v lithium dodecyl sulfate (LiDS), 0.8 M lithium chloride, 40 mM EDTA and 8 mM DTT; LBB). The cell lysate was thoroughly pipetted with 200 µl tip until the sample viscosity was reduced, and pipetting was seamless. 500 µl of oligo(dT)25 magnetic beads (New England BioLabs) suspension was used per replicate. The beads were washed with 1 ml of LBB twice, each time collecting the beads on a magnet and completely removing the supernatant. Upon washing, the oligo(dT)25 beads were resuspended in the cell lysate and placed in a rotator set for 20 rpm at 25 °C for 5 minutes, followed by the same rotation at 4 °C for 30 minutes. The suspension was briefly spun down at 12,000 g, separated on a magnet, and the supernatant was discarded. The beads were then resuspended with 1 ml of wash buffer (20 mM Tris-HCl pH 7.4, 0.2 % v/v Titron X-100, 0.4 M lithium chloride, 10 mM EDTA and 8 mM DTT; WB) and washed on a rotator set for 20 rpm at 4 °C for 5 minutes, using 3 rounds of washing. The beads were collected on a magnetic rack and the supernatant was discarded. The wash procedure was repeated three times. The elution was carried out stepwise. Washed bead pellet was first resuspended in 50 µl of the elution buffer (25 mM HEPES-KOH, 0.1 mM EDTA; HE). The first suspension was heated at 60 °C for 5 minutes to facilitate the elution, and the eluate was collected upon placing the bead-sample mixture on a magnetic rack, separating the beads, and recovering the clean supernatant. The resultant pellet was next resuspended in another 50 µl of HE buffer, and the process was repeated. The eluates were then combined and subjected to an additional solid phase reversible mobilization (SPRI) bead purification step and stored frozen. The eluate from oligo(dT) bead extraction was further purified using AMPure XP SPRI beads (Beckman Coulter Life Sciences) according to the manufacturer's recommendations. Briefly, the eluate samples were supplemented with 1.2x volumes of the SPRI bead suspension in its standard (supplied) binding buffer, and the resultant mixture incubated at room temperature for 5 minutes with periodic mixing. The SPRI beads were brought down by a brief 2,000 g spin and separated from the solution on a magnetic rack. The supernatant was removed, and the beads were resuspended in 1 ml of 80 % v/v ethanol, 20 % v/v deionized water mixture and further washed by tube flipping. The bead and solution separation procedure were repeated. The ethanol washing process was repeated one more time. Any remaining liquid was brought down by a brief spin and removed using a pipette, and the beads were allowed to air-dry while in the magnetic rack for 2 minutes. The purified RNA was then eluted in deionized water and the RNA content was assessed using absorbance readout via Nanodrop and fluorescence-based detection via Qubit RNA high sensitivity (HS) assay kit (Thermo Fisher Scientific).  The library preparation followed the manufacturer's recommendations. 650-800 ng from HeLa cells, were used for each 2× library preparation within every replicate (all recommended volumes doubled-up) with direct RNA sequencing kit (SQK-RNA002) as supplied by Oxford Nanopore Technology. The modifications were that Superscript IV RNA Polymerase (Thermo Fisher Scientific) was used, RNA Control Standard (RCS) was omitted, and RNasin Plus (Promega) was included at 1 U/ µl in all reaction solutions until the SPRI purification step after the reverse transcription reaction. The final adaptor-ligated sample was eluted in 40 µl. 