title: GSM3453187: A12_low-dose antibiotics; Sus scrofa; RNA-Seq
library_construction_protocol: For RNA-seq:RNAs were extracted by Trizol and quantified by Qubit®3.0 fluorometer (Thermo fisher scientific, Cat#Q33216). A total amount of 1.5 μg RNA per liver sample was used as input material for preparations. For Bisulfite-seq:DNAs of liver tissues from these piglet samples were extracted by DNeasy Blood & Tissue Kits (QIAGEN, Cat#69504) according to the manufactural instruments and quantified by Qubit®3.0 fluorometer (Thermo fisher scientific, Cat#Q33216). For RNA-seq:Sequencing libraries were constructed using NEBNext® UltraTM RNA library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations. In brief, mRNA was purified from the total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5×). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH). Second strand cDNA synthesis was subsequently performed using DNA polymerase I and RNaseH, with the dUTP instead of deoxythymidine triphosphate (dTTP). Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. To select cDNA fragments with right length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR, which allowed selective degradation of the second cDNA strand contained dUTP for the reservation of the transcript direction and strand-specific information. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using HiSeq4000 PE Cluster Kit (Illumina) according to the manufacturer's instructions. For Bisulfite-seq: Briefly, 1 μg DNA of per sample was processed by fragmentation using Covaris, following with blunt end repair, 3′-adenylation, and 5′-methylcytosine index adapter ligation. Then, 250 ng DNA from each of three/four adapter-ligated libraries were pooled together for the liquid hybridization capture procedure. The hybridization probe was synthesized and purchased from Roche Nimblegen Incorporation. Finally, the captured DNA was eluted in 50 μl of 10 M NaOH with incubation at room temperature for 10 min. The supernatant was transferred into a new tube and neutralized with 50 μl of 10 M HAc and then purified using MiniElute PCR Purification Kit (QIAGEN, Cat#28004). For bisulfite conversion, 200 ng of unmethylated λDNA was added into each captured product and then ZYMO EZ DNA Methylation-Gold Kit™ (ZYMO, Cat#D5005) was employed to convert unmethylated cytosine into uracil according to the instructions. After purification, PCR was carried out with JumpStart™ Taq DNA Polymerase (Sigma, Cat#D9307) using the program of 94°C 30s, 15 cycles of 94°C 10 s, 60°C 30 s, 72°C 40s then prolong at 72°C for 5 min and hold at 12°C. The PCR products were purified using AMPure XP beads (Agencourt, Cat#A63881) and were quantified by the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
attributes.tag: GEO Accession
attributes.value: GSM3453187