title: GSM7110920: Dysplasia [MK_47]; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA integrity was verified using RNA 6000 Pico Assay chips run on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). To deplete ribosomal RNA, 100 ng of total RNA from each sample was incubated with RiboErase Mouse rRNA depletion oligos per the manufacturer's instructions using Kapa RiboErase (HMR) Module (Kapa Biosystems, USA). Resulting rRNA duplexed with DNA oligos were digested with RNase H treatment. Libraries were then prepared using Kapa RNA HyperPrep Library Preparation Kit according to the manufacturer's protocol. Briefly, after rRNA depletion, the RNA was fragmented at 94C for 6 minutes. First strand cDNA was generated, and dUTP was incorporated during synthesis of second strand cDNA. Next, double-stranded cDNA underwent A-tailing, adaptor ligation, and incorporation of sample-specific multiplex indices before PCR amplification (12 cycles) according to the manufacturer's protocol (Kapa Biosystems, USA). Size distribution and molarity of amplified cDNA libraries were assessed via the Bioanalyzer High Sensitivity DNA Assay (Agilent Technologies, USA). All cDNA libraries were sequenced on an Illumina NextSeq 500 instrument with 1.45 pM input and 1% PhiX control library spiked in (Illumina, USA) targeting 30-40 Million reads pairs per sample.