15 protocols
AccessionType
normalization data transformation protocol
We used the htseq-count Python script to determine the number of reads from each BAM file that mapped to each feature within the GRCm38 release 70 GTF file. The script was run in ╥union╙ mode so that reads overlapping more than one feature were ignored. The read counts generated by this procedure were used as input to version 2.13 of the DESeq2 package for the R statistical computing environment (version 3.0.2). To permit comparison between samples and to visualise gene expression, the counts for each sample were normalised by the size factor for that sample as calculated by DESeq2. Expression levels were then divided by gene length to give size-factor adjusted, length-normalised counts or were transformed using the regularised-log (rlog) transformation available in DESeq2.
high throughput sequence alignment protocol
Reads were mapped using the spliced-read mapper GSNAP version 2013-10-255. Reads were aligned against the GRCm38 mouse genome build release 70 downloaded from Ensembl. GSNAP command line parameters were ╥-A sam -B 5 -t 10 -n 1 -Q nofails so that only uniquely-mapped reads were reported.
nucleic acid sequencing protocol
Single-end, 36bp reads.
growth protocol
CD4+, CD62L+ cells were purified from DEREG mice with CD4+CD62L+ T Cell Isolation Kit II (Miltenyi Biotec). Cells were seeded into anti-CD3 (1 ug/ml, clone 145-2C11, eBioscience) and anti-CD28 (5 ug/ml, clone 37.51, eBioscience) coated 96-well plates, at a density of 250,000-500,000 cells/ml and cultured in a total volume of 200 ul in the presence of recombinant human TGF-beta1 (20ng/ml, Sigma). The cells were gently removed from the activation plate on day 4. The iTreg culture was subjected to FACS sorting for GFP-positive, propidium iodide-negative cells.
nucleic acid library construction protocol
RNA was purified, reverse transcribed and prepared for sequencing described in http://msb.embopress.org/content/7/1/497
nucleic acid sequencing protocol
Paired-end, 100bp reads
growth protocol
CD4+, CD62L+ cells were purified with CD4+CD62L+ T Cell Isolation Kit II (Miltenyi Biotec) and further purification was performed by FACS using anti-CD62L and anti-CD4 Fab fragments.
nucleic acid extraction protocol
Poly-(A)+ RNA was purified from ~500,000 cells using the Oligotex kit (Qiagen). The manufacturers protocol was modified slightly to include additional final elution steps resulting in a larger volume.
nucleic acid sequencing protocol
Paired-end, 36bp reads
growth protocol
MACS was used to isolate splenic CD4+ T cells by depleting CD8a, CD11b, CD11c, CD19 Ly6G(Gr-1) expressing cells followed by positive selection for CD25+ cells.
growth protocol
CD4+, CD62L+ cells were purified with CD4+CD62L+ T Cell Isolation Kit II (Miltenyi Biotec) and further purification was performed by FACS using anti-CD62L and anti-CD4 Fab fragments. Cells were seeded into anti-CD3 (1 ug/ml, clone 145-2C11, eBioscience) and anti-CD28 (5 ug/ml, clone 37.51, eBioscience) coated 96-well plates, at a density of 250,000û500,000 cells/ml and cultured in a total volume of 200 ul in the presence of recombinant human IL-6 (30ng/ml, Immunotools), recombinant human TGF-beta1 (5ng/ml, Sigma), recombinant murine IL-1beta (10ng/ml, Immunotools), recombinant murine IL-23 (10ng/ ml, R&D systems), neutralizing anti-IL4 (5ug/ml, clone 11B11, eBioscience), neutralizing anti-IFN-gamma (5ug/ml, clone XMG1.2, eBioscience), and neutralizing anti-IL2 (5ug/ml, clone JES6-5H4, eBioscience). Cells were labelled with anti-CCR6-APC (1:200, clone 29-2L17, BioLegend), anti-CD8a-FITC (1:1500, clone 53-6.7, eBioscience), and propidium iodide. FACS sorting was then performed to select FITC-negative, APC-positive, propidium iodide-negative cells.
nucleic acid library construction protocol
Samples were processed using the TruSeq RNA Sample Prep v2 kit (Illumina) according to the manufacturers instructions with an alteration to use KAPA Hifi polymerase for PCR amplification instead of the one supplied with the kit.
nucleic acid extraction protocol
RNA was purified using the Qiagen RNEasy Plus Mini Kit automated using a QIAcube
growth protocol
CD4+, CD62L+ cells were purified with CD4+CD62L+ T Cell Isolation Kit II (Miltenyi Biotec). Cells were seeded into anti-CD3 (2 ug/ml, clone 145-2C11, eBioscience) and anti-CD28 (5 ug/ml, clone 37.51, eBioscience) coated 96-well plates, at a density of 250,000-500,000 cells/ml and cultured in a total volume of 200 ul in the presence of recombinant murine IL-12 (10 ng/ml, R&D Systems) and neutralising anti-IL4 (10 ug/ml, clone 11B11, eBioscience). Cells were cultured for another four days in absence of CD3 and CD28 stimulation to rest them. The original medium was kept and fresh medium containing the same cytokines as before was added to dilute the cultures 1:3. Finally, the cells were labelled with anti-CXCR3-APC (1:200, clone CXCR3-173, BioLegend) and Propidium Iodide and FITC-conjugated antibodies against CD11b, CD11c, Ly6G, CD8a and CD19. The cultures were then FACS-sorted to obtain Propidium Iodide-, FITC-negative and CXCR3-positive cells.
growth protocol
CD4+, CD62L+ cells were purified with CD4+CD62L+ T Cell Isolation Kit II (Miltenyi Biotec). Cells were seeded into anti-CD3 (2 ug/ml, clone 145-2C11, eBioscience) and anti-CD28 (5 ug/ml, clone 37.51, eBioscience) coated 96-well plates, at a density of 250,000-500,000 cells/ml and cultured in a total volume of 200 ul in the presence of recombinant murine IL-4 (10 ng/ml, R&D Systems) and neutralizing anti-IFN-gamma (10 ug/ml, clone XMG1.2, eBioscience). Cells were cultured for another four days in absence of CD3 and CD28 stimulation to rest them. The original medium was kept and fresh medium containing the same cytokines as before was added to dilute the cultures 1:3. Finally, the cells were labelled with Propidium Iodide and FITC-conjugated antibodies against CD11b, CD11c, Ly6G, CD8a and CD19. The cultures were then FACS-sorted to obtain Propidium Iodide-, FITC-negative cells.