GSM3066049: R8_1wk; Homo sapiens; RNA-Seq
Total RNA was isolated using EZNA PX blood RNA kit (Omega bio-tek), and then concentrated using RNA clean & concentrator-5 (ZYMO research). Alpha- and beta-globin mRNAs were depleted from total RNA samples using GLOBINclear-human globin mRNA removal kit (Life Technologies) according to manufacturer's instruction. 3SEQ libraries were constructed based on previously published method (Beck AH, 2010, https://doi.org/10.1371/journal.pone.0008768) with modifications. Briefly, after globin mRNA reduction, remaining mRNAs were enriched by poly-A selection using Dynabeads mRNA purification kit (Life Technologies), and heat-fragmented to 100-200 bases. First-strand cDNAs were synthesized using Superscript III reverse transcriptase with Rd2SP-oligodT primer, followed by second-strand cDNA synthesis. Adenine was added to the 3'-end of the double-stranded cDNA and then ligated to the P5-R1SP adapter. The ligated product was amplified by PCR for 15 cycles using primers P5-Rd1SP and P7-index-Rd2SP. Six Illumina indexes, each with six bases, were introduced in the P7 primer. Quality of the library was examined using Agilent DNA 1000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies). Libraries were quantified by Qubit 2.0 fluorometer using Qubit dsDNA BR assay kit (Life technologies). Six samples with different indexes were pooled together and submitted for sequencing at Stanford Center for Genomics and Personalized Medicine. Index associated 36 bp sequences from the P5 primer end, providing the 5'-end sequences of the polyA-containing mRNA fragments, were generated using Illumina HiSeq 2000 system (Illumina, San Diego, CA). RNA-Seq, specifically, 3'-end sequencing for expression quantification (3SEQ)