GSM9045347: MCOLN3 Y391D #2; Homo sapiens; RNA-Seq
Total RNA was isolated from cells using the RNeasy Plus Mini kit (Qiagen; Cat#: 74134) and eluted in nuclease-free H2O. Poly(A) Tail RNA Selection 250-300 bp Insert cDNA Library Strand-Specific Preparation by LC Sciences. Library construction and sequencing is described by LCScience.com as: "Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure. The total RNA quality and quantity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number >7.0. Approximately 10 ug of total RNA was subjected to isolate Poly (A) mRNA with poly-T oligo attached magnetic beads (Invitrogen). Following purification, the poly(A) mRNA fractions is fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with a strand-specific library preparation by dUTP method. The average insert size for the paired-end libraries was 300±50 bp. And then we performed the paired-end 2×150bp sequencing on an Illumina Hiseq 4000 at LC Sciences following the vendor's recommended protocol.