Anti mouse cd3 cd28 dynabeads

CD8+ T cells were purified from Hif1an^fl/fldLck Cre- or Vhl^fl/fldLck Cre- (‘WT’), Hif1an^fl/fldLck Cre+ (‘FIH KO’), Vhl^fl/fldLck Cre+ (‘VHL KO’) and Hif1an^fl/flVhl^fl/fldLck Cre+ (‘FIH/VHL KO’) mice spleens using Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-117-044), followed by magnetic bead isolation on a MACS column. CD8+ T cells were activated with anti-mouse CD3/CD28 Dynabeads (Thermo Fisher) at a 1:1 cell/bead ratio. After 3 days of culture, Dynabeads were removed using a magnet and cells were washed twice with ice cold PBS and lysed with RLT buffer containing 1% β-mercaptoethanol. Total RNA was subjected to quality control with Agilent Tapestation according to the manufacturer’s instructions. To construct libraries suitable for Illumina sequencing the Illumina TruSeq Stranded mRNA Sample preparation protocol which includes cDNA synthesis, ligation of adapters and amplification of indexed libraries was used. The yield and quality of the amplified libraries were analysed using Qubit by Thermo Fisher and the Agilent Tapestation. The indexed cDNA libraries were normalised and combined and the pools were sequenced on the Nextseq 550 for a 50-cycle v2.5 sequencing run generating 2x75 bp paired-end reads. The raw RNA-seq data are deposited in the GEO database under the series number GSE234381.

CD8+ T cells were isolated from mouse spleens and purified using Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-117-044), followed by magnetic bead isolation on a MACS column. CD8+ T cells were activated with anti-mouse CD3/CD28 Dynabeads (Thermo Fisher) at a 1:1 cell/bead ratio. After 3 days of culture, Dynabeads were removed using a magnet and cells were prepared for flow cytometry analysis or cultured for a further 4 days. OT-I CD8+ T cells from OT-I mouse spleens were activated with anti-mouse CD3/CD28 Dynabeads or with 1μg/ml SIINFEKL peptide for 48 hours. Following this, OT-I T cells were washed twice with PBS and cultured for a further 5 days. All T cells were cultured in complete RPMI medium containing 2mM glutamine, supplemented with 10% FBS, 1% Streptomycin, 55μM β-mercaptoethanol (all Thermo Fisher) and 100 units/ml recombinant human IL-2 (Roche, 10 799 068 001).

CD8+ T cells were isolated from mouse spleens and purified using Microbeads conjugated to monoclonal anti-mouse CD8α (Ly-2; isotype: rat IgG2a) antibody (Miltenyi, 130-117-044), followed by magnetic bead isolation on a MACS column. CD8+ T cells were activated with anti-mouse CD3/CD28 Dynabeads (Thermo Fisher) at a 1:1 cell/bead ratio. After 3 days of culture, Dynabeads were removed using a magnet and cells were prepared for flow cytometry analysis or cultured for a further 4 days. OT-I CD8+ T cells from OT-I mouse spleens were activated with 1μg/ml SIINFEKL peptide for 48 hours. Following this, OT-1 T cells were washed twice with PBS and cultured for a further 5 days. All T cells were cultured in complete RPMI medium containing 2 mM glutamine, supplemented with 10% FBS, 1% Streptomycin, 55 μM β-mercaptoethanol (all Thermo Fisher) and 100 units/ml recombinant human IL-2 (Roche, 10 799 068 001).