GSM9047778: hPSC, shPIN1, TGFB, rep2; Homo sapiens; RNA-Seq
Qiagen Rneasy kit For paired end RNA-seq, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library then underwent end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and RT-PCR for quantification and bioanalyzer for size distribution. Quantified libraries were pooled and sequenced on an Illumina NovoSeq X plus. For single end RNA-seq, sequencing libraries were constructed using a TruSeq Stranded mRNA kit (Illumina, San Diego, CA). Briefly, poly(A)+ RNA was isolated using oligo-dT-coated magnetic beads. The recovered RNA was then chemically fragmented. First strand cDNA was generated using random hexamers as primers for reverse transcriptase. Strand-specific second strand cDNA synthesis was performed and a single 'A' nucleotide was then added to each end. Illumina adaptors were ligated to the cDNAs. Limited-round (15-cycle) PCR was used to amplify the material to yield the final libraries. Library concentration was determined using real-time PCR with primers complementary to the Illumina adaptors. Sample libraries were diluted and applied to an IlluminaNextSeq500 HiOutput flow cell. Llibraries were prepared with indexing barcodes to permit multiplexing in a single lane. The 75-cycle single read sequencing was used to assemble the reads into standard fastq formatted data.