title: GSM9328837: NT-shRNA_control_monomac6_1; Homo sapiens; OTHER
library_construction_protocol: Cellular total RNA in TRIzol LS (ThermoFisher #10296028) was extracted by Direct-zol RNA Miniprep kit (Zymo #R2070) or by Phenol Chloroform extraction. mRNA was purified by Dynabeads Oligo (dT)25 (Ambion #61002). Library strategy: Direct-RNA Nanopore-seq; RNA sequencing libraries were prepared from total RNA or mRNA using the Nanopore Direct RNA Sequencing kit (ONT, SQK-RNA004) according to the manufacturer's instructions, with some notable adjustments. Samples were multiplexed following the SeqTagger method from Pryszcz et al. 202523 using the custom RT adapters for the b04_RNA004 model (see Table S4). In brief, 500 ng total RNA or 100 ng polyA-selected RNA in 8 µl nuclease-free water were barcoded with 1µl custom RT adapters via ligation with 1.5 µl T4 DNA ligase (NEB, M0202L), 3 µl 5x NEBNext Quick Ligation reaction buffer (NEB, B6058S), 0.5 µl diluted RCS, and 1 µl SUPERase·In RNase Inhibitor (ThermoFisher #AM2696) for 15 min at room temperature. Samples were then reverse transcribed with 1 µl Induro Reverse Transcriptase (NEB, M0681L), 8 µl 5x Induro buffer, 2 µl 10 mM dNTP, and 14 µl nuclease-free water following the incubation protocol: 55°C for 50 min, 70°C for 10 min, and 12°C hold. RNA-cDNA products were cleaned up with 1.8X AMPure XP Beads (Beckman, A63881) and washed twice in 70% ethanol before eluting in water and pooling the 4 barcoded libraries together for a total of 20 µl. Next, the pooled library was ligated with 6 µl RNA ligation adapter (RLA), 3 µl T4 DNA ligase, 8 µl 5X NEBNext Quick Ligation buffer, and 3 µl nuclease-free water, incubating for 10 min at room temperature. The ligation product was cleaned up with 0.8X AMPure XP beads, washed twice with the provided wash buffer (WSB), and eluted in 33 µl RNA elution buffer (REB). Each library was quantified using 1 µl for Qubit 1x dsDNA HS assay (ThermoFisher, Q33231), with a recovery aim greater than 30ng total. Samples were loaded onto primed MinION (ONT, FLO-MIN004RA) or PromethION flow cells (ONT, FLO-PRO004RA) and sequenced on a MinION or PromethION for 2-22 hours, acquiring 150 Mb to 7.6 Gb (see Table S1 for details). Flow Cell Nuclease Flushing: For Flow Cells which were washed and reused, a modified Flow Cell wash mix was prepared23 with 20 µl TURBO DNase (2U/µl) (Thermo Fisher, AM2238) and 380 µl DIL (wash dilutent, provided with ONT EXP-WSH004). Wash mix was loaded onto Flow Cell and incubated for 20 min before storing or loading another library.