title: lrRNA-Seq of Lung adenocarcinoma cell line
design: Direct RNA (SQK-RNA004) libraries were prepared using 2ug of total RNA with Sequins and SIRV spike-ins. Induro Reverse Transcriptase (NEB, M0681S) was used for reverse transcription for 1 hour 30 minutes at 50C. Approx. 200ng of final library per sample was loaded onto RNA flowcells (FLO-PRO004RA). RNA flowcells were washed with ~1500-2000 active pores remaining using a custom nuclease flush protocol, including 2ul WMX (ONT), 1ul RNase Cocktail (Invitrogen, AM2286), 1ul RNase H (NEB, M0297S). Freshly prepared library for each sample was then reloaded to complete sequencing. Samples were later subjected to further sequencing on new flowcells to achieve bulk-level read depth.