GSM6031105: MFGE8_WT_3; Mus musculus; RNA-Seq
RNA from primary enterocytes were isolated using Qiagen RNeasy plus micro kit. RNA from small intestinal tissues were isolated using Qiagen RNeasy lipid tissue mini kit. Barcoded sequencing libraries were prepared using the QuantSeq FWD kit (Lexogen, Vienna, Austria) for multiplexed sequencing according to the recommendations of the manufacturer using the UDI-adapter and UMI Second-Strand Synthesis modules (Lexogen).  High integrity total RNA samples were processed according to the QuantSeq default protocol. The fragment size distribution of the libraries was verified via micro-capillary gel electrophoresis on a LabChip GX system (PerkinElmer, Waltham, MA).   The libraries were quantified by fluorometry on a Qubit fluorometer (LifeTechnologies, Carlsbad, CA), and pooled in equimolar ratios. The library pool was Exonuclease VII (NEB, Ipswich, MA) treated, SPRI-bead purified with KapaPure beads (Kapa Biosystems / Roche, Basel, Switzerland) , quantified via qPCR with a Kapa Library Quant kit (Kapa Biosystems) on a QuantStudio 5 RT-PCR system (Applied Biosystems, Foster City, CA). Up to 48 libraries were sequenced per lane on a HiSeq 4000 sequencer (Illumina, San Diego, CA) with single-end 100 bp reads.