GSM5799974: LN_Mannan_32; Mus musculus; RNA-Seq
GSM5799974
RNA was isolated from TRI Reagent samples using phenol-chloroform extraction or column-based extraction systems (Direct-zol RNA Microprep and Miniprep, Zymo Research) according to the manufacturer's protocol. RNA concentration and purity (260/280 and 260/230 ratios) were measured by NanoDrop (Thermo Fisher Scientific). RNA samples were quantified using Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and RNA integrity was checked with RNA Screen Tape on Agilent 2200 TapeStation (Agilent Technologies). RNA sequencing library preparation was prepared using TruSeq Stranded mRNA library Prep kit following manufacturer's protocol (Illumina, Cat# RS-122-2101). Briefly, mRNAs were first enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 8 minutes at 94°C. First strand and second strand cDNA were subsequently synthesized. The second strand of cDNA was marked by incorporating dUTP during the synthesis. cDNA fragments were adenylated at 3'ends, and indexed adapter was ligated to cDNA fragments. Limited cycle PCR was used for library enrichment. The incorporated dUTP in second strand cDNA quenched the amplification of second strand, which helped to preserve the strand specificity. Sequencing libraries were validated using DNA Analysis Screen Tape on the Agilent 2200 TapeStation (Agilent Technologies), and quantified by using Qubit 2.0 Fluorometer (Thermo Fisher Scientific) as well as by quantitative PCR (Applied Biosystems). The sequencing libraries were multiplexed and clustered on 1 lane of flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq instrument according to manufacturer's instructions. The samples were sequenced using a 2x150 Pair-End (PE) High Output configuration. 