title: GSM7245766: AU-565 (TEQUILA-seq, blocking oligos(+), multiplex(+)); Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA of breast cancer cell lines was extracted using TRIzol reagent (Invitrogen, #15596018). A 200 ng aliquot of total RNA was used as templates for cDNA synthesis following the SMART-seq2 protocol. 500 ng of amplified cDNA were used for hybridization and capture following TEQUILA-seq protocol with blocking oligos included. The final nanopore 1D library waw constructed using 200 to 500 ng of post-capture amplified cDNA according to the SQK-NBD114.24 protocol. Briefly, cDNA products were end-repaired and dA-tailed using NEBNext Ultra II End Repair/dA-Tailing Module (NEB, # E7546) by incubating at 20°C for 20 min and 65°C for 20 min. The cDNA was then purified with 1× volume of AMPure XP beads and eluted in 10 μl of nuclease-free water. Native barcode ligation was performed using 7.5 μl of end-prepped DNA, 2.5 μl of native barcodes and 10 μl of Blunt/TA Ligase Master Mix (NEB, #M0367) by incubating at room temperature for 20 min. To inactivate the ligase, 2 μl of EDTA was added to each ligation reaction. Barcoded cDNA samples were then pooled and purified using 0.4× volume of AMPure XP beads and eluted in 35 μl of nuclease-free water. Next, adapter ligation was performed using 30 μl of pooled barcoded samples, 5 μl of native adapter, 10 μl of 5× NEBNext Quick ligation reaction buffer and 5 μl of NEBNext Quick T4 DNA ligase (NEB, #E6056) at room temperature for 20 min. After ligation, libraries were purified using 0.45× volume of AMPure XP beads and short fragment buffer.