Name: GSM8206992
Instrument: Illumina NovaSeq 6000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Total RNA was extracted using the Trizol reagent (Thermo Fisher, CA, USA, cat. 15596018) following the manufacturer's procedure. The total RNA quantity and purity were analyzed with Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA, cat. 5067-1511); high-quality RNA samples with RIN number >7.0 were used to construct the sequencing library. After total RNA was extracted, mRNA was purified from total RNA (5ug) using Dynabeads Oligo (dT) (Thermo Fisher) with two rounds of purification. Following purification, the mRNA was fragmented into short fragments using divalent cations under elevated temperature [Magnesium RNA Fragmentation Module (NEB, USA, cat. e6150) under 94℃, 5-7 min]. Then the cleaved RNA fragments were reverse transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, USA, cat. 1896649), which were next used to synthesize U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat. m0209), RNase H (NEB, cat. m0297) and dUTP Solution (Thermo Fisher, cat. R0133). An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat labile UDG enzyme (NEB, cat. m0280) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec and then final extension at 72℃ for 5 min. The average insert size for the final cDNA libraries was 300±50 bp. In the end, we performed the 2×150 bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 following the vendor's recommended protocol. A cDNA library constructed by technology from the pooled RNA from murine species was sequenced and run with Illumina NovaseqTM 6000 sequence platform. Using the Illumina paired-end RNA-seq approach, we sequenced the transcriptome, generating a total of million 2 x 150 bp paired-end reads. The reading obtained from the sequencing machine include raw reads containing adapters or low-quality bases which will affect the following assembly and analysis.