GSM5182604: LUC-LUC rep4; Rattus norvegicus; RNA-Seq
At the time of collection (DIV6), cells were washed with ice-cold Hank's Balanced Salt Solution (HBSS; Thermo Fischer) and lysed with TRIzol reagent (#15596026, Thermo Fisher) by scrapping. The reaction was allowed to proceed on ice for 5 minutes, at which point samples were centrifuged at 12,000 g and 4ºC and supernatants transferred to new tubes before a 1:1 (v/v) dilution with molecular biology-grade 100% ethanol (Millipore Sigma). The RNA extraction protocol was continued using the Direct-zol RNA MicroPrep kit (Zymo Research), in accordance to manufacturer's instructions, and included an on-column DNA digestion step with DNase I. RNA was typically eluted in 30 ul of water. RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina following manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. The sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). RNA-seq. Samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS).
GEO
GSM5182604