GSM6858656: xmoo1_line_ctrl_3_reseq; Homo sapiens; OTHER
Organoids were dissolved in Trizol und RNA was extracted in bulk. Ribosomal RNA was depleted using Rnase H (Epicentre) For generation of full-length cDNA libraries, polyadenylated RNA molecules were isolated from total RNA preparations using the NEB's NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490, NEB). Purified polyadenylated RNA molecules were used for nanopore library preparation using the cDNA-PCR Sequencing protocol (SQK-PCS109, Oxford Nanopore was used). The following modifications were made to the procedure. To eliminate short reads from the final data both input polyadenylated RNA molecules and cDNA molecules were cleaned upon further processing using AMPure XP beads (A63881, Beckman&Coulter) using a magnetic bead sample ratio of 0.4. To exclusively retain cDNA fragments > 3kb the BluePippin device and appropriate separation DNA gel cassettes were used (sage science). Finally, the cDNA was amplified using 14 PCR cycles and 12 min extension time at 65°C. Libraries were sequenced on GridION sequencing device from Oxford Nanopore Technologies. Reads were processed using the pipeline described in: git@github.com:LeilyR/nanoporeReads_dataTransfer.git​ (For cDNA data: guppy-5.0.7 model: dna_r9.4.1_450bps_sup.cfg was used). cDNA-PCR Sequencing protocol (SQK-PCS109)