title: GSM7152349: Ileum, 3 days after first switch, sample #1; Mus musculus; RNA-Seq
library_construction_protocol: Peyer's patches (PPs) were removed from small intestine (SI) and put in TriFastTM (PeqLab, Cat.Nr.: 30-2010). Small intestine was divided into duodenum, jejunum and ileum and each segment was put in TriFastTM. Total RNA was purified using the NucleoSpin RNA II kit (Macherey & Nagel), according to manufacturer's instructions. RNA sequencing was performed on total PPs, ileum or FACS-sorted CD4+Foxp3- or CD45.1+ OT-II viable T cells isolated from PPs or dLN, respectively For library preparation of total PPs and ileum samples, cDNA was used as input to construct 250~300 bp insert cDNA libraries using NEBNext® UltraTM RNA Library Prep Kit (New England Biolabs, Ipswich, USA). Indices were included to multiplex multiple samples. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. For FACS-sorted cells, total RNA was amplified using SMART-Seq™ v4 Ultra™ Low Input RNA Kit for Sequencing (Takara Bio USA, Inc., Mountain View, USA) with the double strand cDNA (ds-cDNA) being synthesized. Then ds-cDNA was purified with AMPure XP beads and quantified with Qubit (Life TechnologiesTM).  The library was ready after end repair, A-tailing, adapter ligation, and size selection. After amplification and purification, insert size of the library was validated on an Agilent 2100 and quantified using quantitative PCR (Q-PCR).