title: GSM5230316: truncated ZMYND11 [aa1-409] 3xFlag-2xstrep in ZMYND11 KO-clone #1; Homo sapiens; RNA-Seq
library_construction_protocol: For long-read RNA-seq, total RNA was extracted with Rneasy mini columns (QIAGEN), TURBO Dnase treated (ThermoFisher Scientific) and poly(A) enriched with NEBNext poly(A) mRNA Magnetic Isolation Module (NEB). Libraries were prepared according to Oxford Nanopore Technologies' instructions for the ONT SQK-DCS109 kit, with each sample barcoded with the ONT EXP-NBD104 kit. Briefly, poly(A) RNA was reverse-transcribed into cDNA with the Maxima H Minus RT (ThermoFisher Scientific) and second strand synthesis was accomplished using LongAmp Taq polymerase (NEB). The blunt DNA ends were prepared using the NEBNext Ultra II End Repair/dA-Tailing module (NEB), adding a 3' dA tail and phosphorylating the 5' end. Barcodes were ligated to the DNA fragments with a Blunt/TA ligase (NEB) and motor protein adapters were finally ligated with the NEBNext Quick Ligation Module (NEB). Purification of DNA between each step was performed with KAPA Hyperpure Beads (Roche). Libraries were sequenced on ONT's MinION Mk1B using four R9 flowcells.