GSM8207960: C1; Mus musculus; RNA-Seq
The RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA).  Subsequently, the RNA integrity was evaluated with the Bioanalyzer 2100 (Agilent, CA, USA).  Poly (A) RNA was isolated using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA) and then fragmented into smaller pieces using the Magnesium RNA Fragmentation Module (NEB, cat. e6150, USA).  The cleaved RNA fragments were reverse-transcribed to generate cDNA utilizing SuperScript™ II Reverse Transcriptase (Invitrogen, cat.  1896649, USA), followed by the synthesis of U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat. m0209, USA), RNase H (NEB, cat. m0297, USA), and dUTP Solution (Thermo Fisher, cat. R0133, USA).  An A-base was added to the blunt ends of each strand to facilitate ligation to the indexed adapters.  Each adapter possessed a T-base overhang for adapter ligation to the A-tailed fragmented DNA.  Single- or dual-index adapters were ligated to the fragments, and size selection was carried out using AMPureXP beads.  Following treatment with the heat-labile UDG enzyme (NEB, cat. m0280, USA) of the U-labeled second-stranded DNAs, the ligated products were amplified via PCR.  The average insert size for the final cDNA library was 300±50 bp.  Finally, 2×150bp paired-end sequencing (PE150) was performed on an Illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) RNA lilbraries for RNA-seg were prepared using SMARTER mRNA-Seg Library Prep Kit