GSM4279372: 5EU 60 min replicate 4; Homo sapiens; RNA-Seq
Direct RNA nanopore sequencing library kit (SQK-RNA001, Oxford Nanopore Technologies) Nanopore: Total RNA was extracted from K562 cells using QIAzol (Quiagen) according to manufacturer's instructions. Poly(A) RNA was purified from 1 mg of total RNA using the µMACS mRNA Isolation Kit (Milteny Biotec) following the manufacturer's protocol. The quality of poly(A) RNA selection was assessed using the TapeStation System (Agilent). Poly(A) selected RNAs were subsequently subjected to direct RNA nanopore sequencing library preparation (SQK-RNA001, SQK-RNA002, Oxford Nanopore Technologies) following manufacturer's protocol with 1000 ng input. All libraries were sequenced on a MinION Mk1B (MIN-101B) for 48 h, unless reads sequenced per second stagnated dramatically. RNA-seq: Total RNA was extracted from K562 cells using QIAzol according to manufacturer's instructions except for the addition of 150 ng RNA spike-in mix 25 together with QIAzol. To isolate polyA RNA from 75 µg of total RNA, two subsequent rounds of purification by Dynabeads Oligo (dT)25 (invitrogen) were performed. Purification based on manufacturer's instructions was performed twice, using 1 mg of Dynabeads Oligo (dT)25 beads for the first round and 0.5 mg for the second round of purification. The quality of polyadenylated RNA selection was assessed using RNA ScreenTape on a TapeStation (Agilent). Sequencing libraries were prepared using the NuGEN Ovation Universal RNA-seq kit according to manufacturer's instructions. Fragments were amplified by 10 cycles of PCR, and sequenced on an Illumina NextSeq 550 in paired-end mode with 75 bp read length.
GEO Accession
GSM4279372