GSM8567357: upperbands barcode 5 H1299 cells; Homo sapiens; RNA-Seq
RNA was extracted (Qiazol) from four human cell lines (iPSCs CHiPs 22 Takara, H1299, Calu-6, A549). cDNAs were synthesized (SuperScript IV Thermo Fisher). Sequencing libraries were prepared following ONT kit instructions with TERT specific primers and ONT sequences with a total 30 cycles. DNA bands in the 2.5 kb – 4 kb range (upper bands; upper TERT) and bands below 2.5 kb to 0.5 kb (lower bands; lower TERT) were excised and purified via gel extraction (Qiagen). Next, size binned fractions were amplified with ONT barcoded primers 1-12 (4 cells lines by 3 replicates by 2 size bins) for 20 cycles. Barcoded libraries from each cell line were pooled at equal molar ratios for each size bin (upper bands or lower bands). The pooled libraries for each size bin were sequenced on its own flow cell (two R9.4.1 flowcells). Sequencing was performed with the standard 72 h sequencing protocol run on the MinION Mk1C, using the MinKNOW software (v 3.3.2). From lower TERT, 21.8 million reads were obtained and 16.4 million reads passed from all 12 samples. From upper TERT, 14.1 million reads were obtained, and 12.2 million reads were passed from all 12 samples. Base-calling was performed by Guppy (v 3.1.5). Fastq files which were classified as passed (Phred score of 8) by the MinKNOW software were subsequently processed and analyzed.