Ovation mouse rna seq system

HpC tissue from mice stereotactically injected with AAV-shRNA-Slc20a1, shRNA-Slc20a2 or Scramble were collected after cervical dislocation. Total RNA was extracted using TRizol reagent (15596026, ThermoFisher Scientific) and RNeasy Mini Kit (74104, QIAGEN) with DNase I treatment step (79254, QIAGEN) following the manufacturer’s protocol. The integrity of RNA was determined by RNA ScreenTape (5067-5576, Agilent Technologies) on the Agilent 2200 TapeStation (Agilent Technologies). RNA-seq libraries were prepared starting from 100 ng of total RNA using the Ovation mouse RNAseq system (Nugen) as recommended by the manufacturer. Ribosomal RNA was depleted by PCR (10 cycles). RNA-seq libraries were sequenced on an Illumina HiSeq2500 (Paired-End sequencing 130 × 130 bases, High Throughput Mode). A minimum of 10 million paired-end reads was produced per library sample. Sequence reads were aligned to the mouse MM38 reference genome using Hisat2 software and counted by featureCounts from the Subread R package. Only unique reads mapped to known transcripts were used for expression analyses; reads mapped to ribosomal RNA, genome and unmapped reads were excluded.

Laser capture microdissection was used to isolate RNA from small intestinal epithelial cells of homeostatic stop^flox and stop^ΔIEC mice. Isolated RNA quality was checked using Picochip kit on 2100 Agilent Bioanalyser. RNA was processed using the Ovation Mouse RNA-seq system (NuGen) to produce a cDNA library. RNA was sequenced on an Illumina platform (1x50bp reads) and data was analyzed using the Galaxy platform [64 (link)]. Briefly, RNA seq reads were analyzed using FastQC and aligned to the mouse genome using Bowtie2. Aligned reads were subjected to differential gene expression analysis using CuffDiff. Top 50 upregulated and downregulated genes (p value < 0.05) were visualized using GraphPad Prism. Raw data is deposited in the NCBI GEO repository (GSE140518). Complete list of differentially regulated genes can be found in S1 Table.