alias: GSM2048403
title: GSM2048403: OTII_CD11b_3; Mus musculus; RNA-Seq
library_construction_protocol: 1. Naive OT-II CD4 T cell extraction: peripheral lymphnodes and spleens were dissected on ice and mechanically disrupted in RPMI/10% FBS. Naïve CD4 T cells were pre-enriched by negative selection using biotinylated antibodies against CD8α, CD25, CD11c, CD11b, TER-119, NK1.1, and B220 and anti-biotin MACS beads (Miltenyi Biotec) and sorted as Aqua–CD11c–CD8α–MHCII–Vα2+CD4+CD25–CD62hiCD44lo. 2. Mesenteric lymphnodes from eight week old male C57Bl6 mice reared in the SPF facility of Rockefeller University were dissected into cold HBSS supplemented with Mg2+ and Ca2+, finely chopped and incubated in 400 U/ml Collagenase D (Roche) in HBSS for 25 min at 37°C, 5% CO2. Collagenase was quenched on ice by addition of final 10% FCS. Single cell suspensions were extracted from connective tissue by taking up and resuspending the digests five times. Mesenteric dendritic cells were pre-enriched using anti-CD11c MACS beads (Miltenyi Biotec) and LS MACS Separation Columns (Miltenyi Biotec). Cells were sorted using FACS Aria cell sorter flow cytometer (Becton Dickinson).Dendritic were sorted as Aqua–CD45+Lin–(CD3–B220–NK1.1–CD19–) CD11chi, and the subpopulations further as MHCIIintCD8α+CD11blo, MHCIIintCD8α–CD11b+, MHCIIhiCD103+CD11b– and MHCIIhiCD103+CD11b+. 3. After 24 h of co-culture with DCs, OT-II cells were sorted as Aqua-CD11c–CD8α–MHCII–Vα2+CD4+. RNA from sorted cell pellets was extracted and column-purified using the Arcturus PicoPure RNA Isolation kit (Applied Biosystems). Genomic DNA was removed by on-column digest with DNase I (Qiagen), according to the PicoPure RNA Isolation kit manual. RNA libraries were prepared using the SMARTer Ultra Low Input RNA for Illumina Sequencing kit (Clontech Laboratories) and sequenced using 50 base pair paired end reading on a HiSeq 2500 instrument (Illumina).
identifiers.namespace: GEO
identifiers.id: GSM2048403