GSM5255692: R1_H1975_ONT; Homo sapiens; RNA-Seq
The mRNA was extracted using a Qiagen RNA miniprep kit and purified using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490). 3.75ng purified mRNA spiked with sequins were used for the NEBNext Ultra II Directional RNA Library Prep Kit (Illumina). Fragmentation was done for 15 minutes at 94°C, and final amplification with 11 PCR cycles. The libraries were sequenced in 75PE on NextSeq 500. The cRNA-PCR Barcoding (SQK-PCS109 with SQK-PBK004) kit was used with the supplied protocol (ONT). Amplication for 1.5ng purified mRNA spiked with sequins was done with 14 PCR cycles with a 2 minutes and 30 seconds extension step. Pooled barcoded libraries were sequenced on 5 PromethION R9 flow cells. Iso-Seq libraries were prepared and sequenced by Novogene on Sequel II (PacBio). Libraries were prepared according to the manufacturer's specifications from total RNA spiked in with sequins. Size selection was performed for the standard ~2kb target. The total number of PCR cycles is 12.