title: GSM7245763: MCF7 (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-LSK109 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 60 μl of nuclease-free water. Adapter ligation was performed using NEBNext Quick T4 DNA ligase (NEB, #E6056) at room temperature for 10 min. After ligation, libraries were purified using 0.45× volumes of AMPure XP beads and short fragment buffer.