GSM2701904: immature liver3; Gallus gallus; RNA-Seq
Total RNA from 3 adult roosters’ and 12 one-day-old rooster’ livers for RNA sequencing were extracted using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer’s procedure. Specifically, the DNaseI was used during the RNA isolation process to avoid contamination with genomic DNA. The quantity and purity of total RNA were analyzed by a NanoDrop® ND-1000 spectrophotometer (Thermo Scientific, MA, USA), and integrity of RNA was accessed with Bioanalyzer 2100 and RNA Nano6000 LabChip Kit (Agilent, CA, USA). Only samples that had the OD260/280 > 1.8, OD260/230 > 2.0, and the RNA Integrity Number (RIN) > 7.0 were used for further sequencing. Four RNA samples of one-day-old chicks’ liver were mixed equally together as a pooled RNA sample according to the purity of total RNA, and the RNA samples of adult chickens’ livers were directly used for library construction. In total, we gathered three RNA samples from three adult roosters and three pooled RNA samples from 12 one-day-old roosters for further library construction. Approximately 3 μg of total RNA was used to prepare an LncRNA library. According to protocol of Epicentre Ribo-zero™ Gold Kit (Illumine, San Diego, USA), ribosomal RNA was removed and the rRNA-depleted RNA (Poly A+ and Poly A－ RNA) were collected [24, 25, 43]. Subsequently, high strand-specificity libraries were generated using the rRNA-depleted RNA and a NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) following the manufacturer’s recommendations. Briefly, the rRNA-depleted RNA was fragmented using divalent cations under elevated temperature in NEBNext. First-strand cDNA was synthesized using random hexamer primers and M-MuLV reverse transcriptase (RNase H−). Subsequently, second-strand cDNA synthesis was performed using second-strand synthesis reaction buffer, DNA polymerase I, and RNase H. Remaining overhangs were converted into blunt ends by exonuclease/polymerase activity. After adenylation of the 3’ ends of the DNA fragments, NEBNext adaptors with hairpin loop structures were ligated to the fragments to prepare them for hybridization. To select cDNA fragments that are 150–200 bp in length, the fragments in each of the library were purified with an AMPure XP system (Beckman Coulter, Brea, CA, USA). Then 3 μl USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. The qPCRs were performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. The PCR products were purified (AMPure XP system) and library quality was assessed on an Agilent Bioanalyzer 2100 system. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using a TruSeq PE Cluster Kit v3-cBot-HS (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. After cluster generation, the paired-end sequencing (2*125bp) were performed on an Illumina Hiseq2500 at the LC-BIO (Hangzhou, China).