title: GSM4514244: Treg_SPF_2-2; Mus musculus; RNA-Seq
library_construction_protocol: The following fluorochrom-conjugated antibodies were purchased from BD, Biolegend and eBioscience (human)-CD2 (RPA-2.10), CD4 (RM4-5), Ova‑TCR (KJ1.26), CD62L (MEL14). To prevent unspecific antibody binding, cell suspensions were incubated with 10µg/ml Chrom Pure rat IgG (Jackson ImmunoResearch Laboratories) and 10 µg/ml anti-mouse CD16/CD32 antibody (BioXcell). For surface staining cells were washed and then incubated with the corresponding antibodies dissolved in PBS containing 0.2% BSA for 15 min on ice. Cell sorting for RNA-seq and ATAC-seq analysis was performed at the cell sorting facility (Helmholtz Centre for Infection Research, Braunschweig, Germany) on FACSAria IIu (BD Bioscience) and FACS Aria-II SORP (BD Bioscience). Acquired data was analyzed utilizing FlowJo software (Tree Star). Foxp3hCD2+CD4+OvaTCR+ Tregs and CD4+OvaTCR+CD62L+ naïve T cells were directly FACS sorted into RLT+ lysis buffer (Qiagen) containing 10 µl/ml β-Mercaptoethanol (Roth). Total RNA was extracted with the RNeasy Plus Micro kit (Qiagen) according to manufactures instructions. cDNA conversion was carried out using the SMART-Seq v4 Ultra Low Input RNA Kit (Takara Clontech Laboratories) according to manufacturer's instructions, followed by PCR product purification with Agencourt AMPure XP magnetic beads (Beckman Coulter). Libraries were prepared with the Nextera XT DNA Library Prep Kit (Illumina) employing enzymatic tagmentation and simultaneous adapter ligation before subsequent indexing and fragment amplification. PCR product clean-up and size selection was performed with the Agencourt AMPure XP magnetic beads. RNA and cDNA quality and content as well as the correct sizing of cDNA library fragments was verified using the Agilent Technologies 2100 Bioanalyzer profiles and Qubit measurements after each step. Sequencing of libraries was carried out at the sequencing facility on Illumina HiSeq2500 using 50 bp single-end reads and quality of sequenced libraries was verified with FastQC.
attributes.tag: GEO Accession
attributes.value: GSM4514244