title: GSM2480735: C57BL/6J_Male_NaiveCD4+Tcell_B6; Mus musculus; RNA-Seq
library_construction_protocol: Naïve CD4+ T lymphocytes were purified from single-cell suspensions by MACS depletion of non-CD4+ T cells as well as regulatory T cells and TCRγ/δ+ T cells, using a cocktail of specific biotin conjugated antibodies against CD8a (Ly-2), CD45R (B220), CD49b (DX5), CD11b (Mac-1), Ter-119, CD25 and TCRγ/δ+ using the CD4+ CD62L+ T Cell Isolation Kit II (Miltenyi) to obtain an unlabeled pre-enriched CD4+ T cell fraction. Collected cells were suspended in PBS containing 2% FBS and Fc receptors were blocked with FcR blocking reagent for 10 minutes at 4°C (Miltenyi). Cells were further fluorescently stained with a combination of CD4-FITC (Miltenyi), CD44-APC clone IM7 (eBioscience), CD62L clone MEL-14 (BD Pharmingen), eFluor® 450 CD25 clone PC61.5 (eBioscience), for 10 minutes at 4°C, washed and then stained with DAPI (Life Technologies) to cell sort the live population of naïve CD4+, CD62Lhi, CD44lo, CD25- T lymphocytes using the MoFlo XDP system (Beckman Coulter). Samples were gated on single cells for doublet discrimination and non-viable cells were excluded using the viability dye 4’, 6 – diamidine – 2 – phenylindole (DAPI). AllPrep DNA/RNA Mini Kit (Qiagen) was used to perform simultaneous isolation of RNA and DNA from 106 sorted naïve CD4+ T cells and resting B cells.  All RNA samples were subjected to in-column RNase-Free DNase treatment (Qiagen) to remove traces of DNA contamination. RNA integrity was subsequently assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies) by using the RNA 6000 Pico Kit (Agilent Technologies). For each sample type 3 biological replicates were generated from male and female samples. AllPrep DNA/RNA Mini Kit (Qiagen) was used to perform simultaneous isolation of RNA and DNA from 106 sorted naïve CD4+ T cells and resting B cells. All RNA samples were subjected to in-column RNase-Free DNase treatment (Qiagen) to remove traces of DNA contamination. RNA integrity was subsequently assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies) by using the RNA 6000 Pico Kit (Agilent Technologies). RNA was extracted from 1 million cells and spiked-in with synthetic ERCC ExFold RNA Spike-In Mixes (Ambion®) according to manufacturer’s instructions. RNA-sequencing libraries were prepared at the Wellcome Trust Sanger Institute using the bespoke pipeline and the TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero Gold (Illumina) following manufacturer’s instructions with minor modifications. 400ng of RNA per sample type were subjected to rRNA-depletion with RiboZero Gold, fragmented for 6 min using metal-ion catalysed hydrolysis. Following the adaptor ligation step an Uracil-Specific Excision enzymatic treatment (NEB) was incorporated to introduce nicks in U positions of the second strand and ensure optimal stranded library preparation. The libraries were amplified with 8 cycles of PCR using the KAPA HiFi HotStart DNA Polymerase (Kapa Biosystems), cleaned up with AMPure XP Beads, quantified and pooled in equimolar amounts. The pools were sequenced at the Wellcome Trust Sanger Institute on the Illumina HiSeq 2000 platform using 100bp paired-end read length.
identifiers.namespace: GEO
identifiers.id: GSM2480735