title: GSM8794132: Tissue, Ighasec-/-, Isotype, Jejunum, ZE5; Mus musculus; RNA-Seq
library_name: GSM8794132
library_construction_protocol: 1 cm of tissue from the middle of the jejunum, the end of the ileum was excised and preserved in RNAprotect (Qiagen) overnight at 4°C. The preserved tissue was then stored at -80°C until processing. Tissue was thawed on ice and transferred to a screw-cap tube containing 450 μL of RLT Plus buffer supplemented with 2-mercaptoethanol and equal quantities of 1.0mm and 0.5 mm zirconium oxide beads (Next Advance). Tissue was homogenized three times for 30 sec at a speed of 6 m/s using Bead Ruptor Elite mill homogenizer (Omni), with 1 min incubation on ice between each cycle. RNA from the lysates was purified using RNeasy Plus Mini kit (Qiagen) with the optional on-column DNase I digest step according to manufacturer protocol. 50000 CD45-EpCAM+ IECs were sorted from the jejunum and the ileum of anti-CD8β, or isotype treated Ighasec+/- and Ighasec-/- mice. Two independent sorting experiments were performed. Cells were collected into RLT Plus buffer (Qiagen) supplemented with 2-mercaptoethanol. RNA was purified using RNeasy Micro kit (Qiagen) and quantified with Power SYBR™ Green RNA-to-CT 1-Step kit. 500 ng of tissue RNA was used as input in the TruSeq Stranded mRNA Library Prep (Illumina). For sorted epithelial cells, the SMART-Seq v4 Ultra Low input RNA kit (Takara) was used to generate cDNA from 10 ng of purified RNA. cDNA was amplified 9 cycles and purified according to manufacturer's protocol. 200 pg of purified cDNA was the used to generate RNA-seq libraries with Nextera XT DNA Library preparation kit (Illumina) according to manufacturer's specifications.