GSM5769605: Human stomach (1D cDNA); Homo sapiens; RNA-Seq
Poly(A)+ RNA (5 μg) from human stomach were purchased from Clontech 50 ng of poly(A)+ RNA from human stomach tissue were used as templates for cDNA synthesis following the SMART-seq2 protocol with some modifications. The reverse transcription and template-switching reaction was performed by Maxima H minus reverse transcriptase (Thermo Scientific, #EP0751) under the following conditions: 42°C for 90 min, 85°C for 5 min. PCR amplification of first-strand cDNA using KAPA HiFi ReadyMix (KAPA Biosystems, #KK2602) was performed by incubating at 95°C for 3 min, followed by 13 cycles of (98°C for 20 s, 67°C for 20 s, 72°C for 4min) with a final extension at 72°C for 5 min. PCR products were treated with Exonuclease I (NEB, #M0293) to remove unused primers and then purified using 0.8X volume of SPRIselect beads (Beckman Coulter, #B23318). 1D Nanopore libraries were then constructed using 1 μg of amplified cDNA according to the standard SQK-LSK109 protocol. 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 minutes and 65°C for 20 min. End-prepared cDNA were purified with 1X volume of AMPure XP beads and eluted in 60 μl of Nuclease-free water. Adapter ligation was then performed using NEBNext Quick T4 DNA ligase (NEB, #E6056) at room temperature for 10 min. After ligation, the libraries were purified using 0.45X volume of AMPure XP beads and short fragment buffer (SFB) to enrich all fragments equally.