title: GSM8037101: BEC cells, R787; Mus musculus; RNA-Seq
library_construction_protocol: Cells were washed once with PBS and total RNA was isolated using the RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. RNA quality was determined using the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). All samples had a RIN ≥ 9.5. 10 µg total RNA for each sample were used for library preparation with the Nanopore Direct cDNA sequencing kit (#SQK-DCS109, Oxford Nanopore Technologies) with native barcode expansions (#EXP-NBD104, ONT) according to manufacturer's protocol (version: DCB_9091_v109_revL_14Aug2019). First, cDNA was synthesized. The ONT primer VNP was incubated for 5 minutes together with the RNA and dNTPs at 65°C and then snap cooled on a pre-chilled block. Afterwards the SSP primer, 5x RT Buffer (supplied with reverse transcriptase) and RNaseOUT™ (#10777019, Thermo Fisher) were added, the samples were mixed by flicking and incubated for 2 minutes at 42°C. Maxima H Minus RT (#EP0753, Thermo Fisher Scientific) was added, samples were mixed and incubated for 90 minutes at 42°C, 5 minutes at 85°C and were then cooled to 4°C. RNA was digested for 10 minutes with RNAse Cocktail Enzyme Mix (#AM2286, Thermo Fisher Scientific) at 37°C followed by cleanup of the samples using AMPure XP beads (#A63881, Beckman Coulter) according to ONT protocol. The eluted cDNA was used for 2nd strand synthesis. To this end, PR2 primer and 2X LongAmp Taq Master Mix (#M0287L, NEB) were mixed with the cDNA samples and incubated for 1 minute at 98°C, 1 minute at 50°C and 15 minutes at 65°C. The double stranded DNA was cleaned up with AMPure XP beads and taken forward to end preparation with the NEBNext® Ultra II End Repair/dA-Tailing Module (#E7546L, NEB). After bead cleanup, native barcode ligation was performed. Each sample was labelled with a single assigned barcode by incubation of the sample together with the barcode and Blunt/TA Ligase Master Mix (#M0367L, NEB) for 10 minutes at 21 °C. After another cleanup step, samples were eluted in 21.7 µl nuclease-free water and pooled. 65 µl of the pooled sample were directly used for adapter ligation, the remaining sample was stored at -20°C. The adapter mix II was ligated to the sample using the NEBNext® Quick Ligation Module (#E6056L, NEB) for 10 minutes at 21°C. During the following bead cleanup, the library was washed with short fragment buffer and eluted in 12 µl elution buffer.