GSM9416004: O4G_ESCs_n2_PCS_R10; Mus musculus; RNA-Seq
Cell lysates were homogenized with QIAshredder columns (Qiagen #79656) and total RNA was extracted using RNeasy Kit (Qiagen #74106) according to the manufacturer's instructions. RNA was quantified using a NanoDropTM 2000 Spectrophotometer (ThermoFisher #ND-2000C) and then DNase-treated using TURBOTM DNase (ThermoFisher Scientific #AM2238) according to the manufacturer's instructions. 300ng of DNase-treated and non-treated RNA were loaded onto a 1% Agarose A (Bio Basic #D0012) gel to verify the RNA integrity and purity. RNA was also quantified using the Qubit™ RNA Broad Range Kit (ThermoFisher Scientific #Q10210) on Qubit™ 4 Fluorometer (ThermoFisher Scientific #Q33238) according to the manufacturer's instructions. RNA quality was further verified using an RNA 6000 Nano Kit (Agilent Technologies #5067-1511) on a 2100 Bioanalyzer instrument (Agilent Technologies #G2939B) according to the manufacturer's instructions. Samples all had RNA integrity numbers of 7.8 or more. For samples sequenced on R9 Flow Cells, polyadenylated RNA was enriched from 7.5-20μg of total RNA using NEB®Next Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) and a DynaMag™-2 Magnet (ThermoFisher Scientific #12-321-D). PolyA+-enriched RNA was quantified using the Qubit™ RNA High Sensitivity Kit (ThermoFisher Scientific #Q32852) on Qubit™ 4 Fluorometer according to the manufacturer's instructions and RNA quality was verified using an RNA 6000 Pico Kit (Agilent Technologies #5067-1513) on a 2100 Bioanalyzer instrument according to the manufacturer's instructions. For samples sequenced on R10 Flow Cells, total RNA was used for RT (Poly(A) enrichment step was omitted). R9 samples were processed with Oxford Nanopore Technologies' (ONT) SQK-DCS109 kit according to the manufacturer's protocol: "Direct cDNA Native Barcoding (SQK-DCS109 with EXP-NBD104 and EXP-NBD114)" version DCB_9091_v109_revF_04Feb2019. 55-100ng of poly(A)+ RNA were used for each sample's RT reaction. Samples were barcoded with the ONT EXP-NBD104 native barcoding expansion. Barcoded samples were pooled, loaded on a MinION sequencer (ONT) using eight R9.4.1 Flow Cells (ONT #FLO-MIN106D), and sequenced using the MinKNOW (v19) software. Purification of DNA between each step was performed with KAPA Hyperpure Beads (Roche). Flow cells were washed and reloaded with the Flow Cell Wash Kit (ONT #EXP-WSH003) to increase sequencing depth obtained per flow cell. R10 samples were processed with ONT's SQK-NBD114.24 kit using 50ng of total RNA, with each sample assigned to a specific barcode. Briefly, an oligo-dT primer (VNP, sequence: 5'-5phos/ACTTGCCTGTCGCTCTATCTTCTTTTTTTTTTTTTTTTTTTTVN-3' , where V=A, C, or G, and N=A, C, G, or T) was used to reverse-transcribe poly(A) RNA into cDNA with the Maxima H Minus RT (ThermoFisher Scientific). A Strand Switching Primer (SSP 5'-TTTCTGTTGGTGCTGATATTGCTmGmGmG-3' , where mG = 2' O-Methyl RNA bases) was used for strand switching during the RT reaction. Libraries were PCR amplified for 14 cycles with LongAmp Taq polymerase (NEB #M0287) or NEBNext Ultra II Q5 Master Mix (NEB #M0544L). cDNA was quantified using the Qubit™ 1X dsDNA High Sensitivity Kit (ThermoFisher Scientific #Q33230) on Qubit™ 4 Fluorometer according to the manufacturer's instructions and cDNA smear quality was verified using a DNA 12000 Kit (Agilent Technologies #5067-1508) on a 2100 Bioanalyzer instrument (Agilent Technologies #G2939B) according to the manufacturer's instructions. 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), barcoded samples were pooled 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). Library was loaded on a PromethION 2 Solo (ONT) sequencer using five R10.4.1 Flow Cells (ONT #FLO-PRO114M), and sequenced using the MinKNOW (v23.07.15) software.