title: GSM9324421: MPC_4,Blood samples from patients with multiple primary cancers _4; Homo sapiens; RNA-Seq
library_name: GSM9324421
library_construction_protocol: For each sample, 500 ng total RNA was transferred to 0.2 ml PCR tubes and adjusted to a final volume of 9 μl with nuclease free water. Reactions were prepared (9 μl total RNA, 1 μl 10 μM VNP primer, 1 μl 10 mM dNTPs) and incubated for 5 min at 65°C, then snap cooled on a pre-chilled freezer block. Strand-switching buffer (4 μl 5x RT buffer, 1 μl RNaseOUT, 1 μl nuclease-free water, and 2 μl 10 μM strand-switching primer) was then added to the snap-cooled, annealed mRNA, and incubated at 42°C for 2 min. 1 μl of Maxima H Minus Reverse Transcriptase was added, and reactions were incubated at 42°C for 90 min, 85°C for 5 min, then held at 4°C. DNA library were set up for each cDNA (2 μl PR1 Primer, 2 μl PR2 Primer, 20 μl first-strand cDNA, 25 μl LongAmp Taq 2x master mix, 1 μl nuclease free water), and cycled for [3 min at 94°C] ×1 cycle, [15 s at 94°C, 15 s at 50°C, 2 min at 65°C] ×12 cycles, [10 min at 65°C] ×1 cycle, then held at 4°C. cDNA were added barcodes using the Native Barcode Kit 96 V14 (SQK-NBD114.96) following manufacturer's guidelines. Each barcoded DNA was purified in 1× Ampure XP Beads, eluted in 20 μl of nuclease free water and quantified using Qubit. Finally, 300 fmol of adapter ligated library was loaded onto a PromethION flow cell. For Long‑read sequencing, the libraries were prepared for following the manufacturer's instructions and applied to MinION system sequencing.
identifiers.namespace: GEO
identifiers.id: GSM9324421_r1