RNA-seq of pig: porcine induced pluripotency stem cell A1 (pips-A1)
A total amount of 2 g RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (E7530L, NEB, USA) following the manufacturers recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from 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 (5X). First strand cDNA was synthesized using random hexamer primer and RNase H. Second strand cDNA synthesis was subsequently performed using buffer, dNTPs, DNA polymerase I and RNase H. The library fragments were purified with QiaQuick PCR kits and elution with EB buffer, then terminal repair, A-tailing and adapter added were implemented. The aimed products were retrieved and PCR was performed, then the library was completed. RNA concentration of library was measured using Qubit RNA Assay Kit in Qubit 3.0 to preliminary quantify and then dilute to 1ng/l. Insert size was assessed using the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA), and qualified insert size was accurate quantification using StepOnePlus Real-Time PCR System (Library valid concentration10 nM). The clustering of the index-coded samples was performed on a cBot cluster generation system using HiSeq PE Cluster Kit v4-cBot-HS (Illumina) according to the manufacturers instructions. After cluster generation, the libraries were sequenced on an Illumina novaseq6000 platform and 150 bp paired-end reads were generated.