GSM5708591: Differentiated enterocytes_CmafIECKO_rep1; Mus musculus; RNA-Seq
Sorted samples were transferred into RLT lysis buffer with β-mercaptoethanol (Qiagen) and snap-frozen on dry ice. RNA was isolated using Qiagen RNeasy Plus Mini Kit (Qiagen). RNA concentration and quality were analysed using 2100 Bioanalyzer (Agilent). RNA-seq libraries were prepared from 500 ng of total RNA. RNA sequencing library preparation used NEBNext Ultra RNA Library Prep Kit for Illumina by following the manufacturer's recommendations (NEB, Ipswich, MA, USA). Briefly, enriched RNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end-repaired and adenylated at 3'ends, and universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. 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 (Applied Biosystems, Carlsbad, CA, USA).  USA). After clustering, the flowcell was loaded on the Illumina HiSeq instrument according to manufacturer's instructions. The samples were 2x150 single-end sequencing using HiSeq 4000 SR, each sample being sequenced on three separate lanes to increase coverage per sample.