GSM3418213: exvivo_rep2_IL15_BNZ; Homo sapiens; RNA-Seq
To isolate RNA from whole duodenal biopsies, a single bioptic fragment was stored in RNA-later (QIAGEN) at the time of endoscopy, kept at 4°C for 24 hours and then frozen at -80°C upon RNA later removal until processing. Defrost tissues were homogenized using magnetic beads and a Cell Tissue Homogenizer (Bullet blender by Next Advance) and RNA was extracted using RNeasy columns (QIAGEN). RNA integrity was assessed by Bioanalyzer (Agilent). 500ng of high-quality RNA was reverse-transcribed into cDNA using the qScript cDNA SuperMix (Quanta Biosciences). TaqMan Gene Expression Assay (Applied BioSystems, final concentration 1X) using Taqman Fast Advanced Master Mix and Taqman specific probes were used to measure the expression levels of IL-15Ra (Hs00542604) and IL-21 (Hs00222327) genes. As housekeeping genes, we used: GAPDH(Hs02758991), GUSB (Hs99999908), HPRT1 (Hs99999909), and POLR2A(Hs00172187). PCR was performed using the following thermal protocol: 50 °C, 2 min; 95 °C, 2 min, PCR Cycle of 40 cycles of (95 °C, 1 sec; 60 °C, 20 sec). After the indicated treatments, cells were collected and homogenized in RLT plus buffer, RNA isolation was carried out using the RNeasy Plus Mini Kit (Qiagen cat#74134) or RNeasy Plus Micro Kit (Qiagen cat#74034) for cell lines and ex vivo samples respectively, according to manufacturer's instructions. RNA quantity was evaluated spectrophotometrically and the quality was assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies); all tested samples showed RNA integrity number (RIN) above 8. RNA-sequencing libraries were prepared using the SMARTer® Stranded Total RNA Sample Prep Kit-HI Mammalian by Clontech Laboratories (Takara) for cell lines and the Illumina TruSeq Total RNA stranded kit (including Ribo-Zero depletion) for ex vivo samples, according to manufacturer's instructions. Libraries quality was checked by Bioanalyzer (Agilent) prior to pooling and sequencing. Indexed cDNA libraries were pooled in equimolar amounts and sequenced with single-end 50bp reads with a high output Flow Cell (8 lane flow cell) on an Illumina HiSeq4000 at the University of Chicago Genomic Facility. In vitro and ex vivo samples were run separately. Number of reads sequenced per sample are in Table S1  .