GSM6523753: Sham_rep3; Rattus norvegicus; RNA-Seq
Total RNA of each sample was extracted by TRK-1002U (LC sciences, Houston, Texas, USA) according to the manufacturer's protocol. The quality and quantity of RNA in each sample was measured by Nanodrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity number (RIN) was assessed by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) with RIN ＞7.0.  approximately 2μg of total RNA from each pooled sample was used to prepare the cDNA library of mRNA by ribosomal RNA depletion method using Ribo-Zero rRNA Removal Kit (Illumina, San Diego, CA, USA). Following purification, the ribo-minus RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 94℃ for 5-7min. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which were next used to synthesise 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 is then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters are ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products are 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 library was 300±50 bp. Library for mRNA, LncRNA, cicRNA was prepared for 2×150bp paired-end sequencing (PE150) on an illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China)  sequencing using standard Illumina protocols.