Macs anti cd8 magnetic beads

An shRNA library focused on 625 epigenetic regulators was cloned into the LENG vector and used to generate a retroviral pool for infection (Cheloufi et al. 2015 (link)). CD8⁺ T cells were isolated from the spleens and lymph nodes of C57BL/6 (WT) mice by MACS anti-CD8 magnetic beads (Miltenyi), activated as described above, and cultured an additional 6 d prior to cell sorting. DNA was extracted by standard methods, and barcoded sequencing libraries were prepared using a nested PCR strategy with AmpliTAQ Gold polymerase (Thermo Fisher): 20 PCR cycles using primers MA389 and MA391 followed by a secondary PCR for 25 cycles with barcoding primers. The resulting libraries were submitted for Illumina SR50 sequencing. Average reads in each of the three replicates corresponding to each shRNA in each fraction were used to determine enrichment.

T cells were isolated from mouse spleen and lymph nodes and purified by positive selection with MACS anti-CD8 magnetic beads according to the manufacturer's protocol (Miltenyi Biotech) or by cell sorting for naïve cells (TCRβ⁺CD8⁺CD25⁻CD62L⁺CD44⁻) on FACSAria (BD Biosciences). For T-cell activation, wells were precoated with 1 mg/mL goat antihamster IgG at 1:40 dilution (MP Biomedicals). T cells were resuspended in T-cell medium (TCM): RPMI (Fisher Scientific) supplemented with 10% FBS (Atlanta Biologicals), 4 mM glutamine, 50 μM β-mercaptoethanol, and 20 mM HEPES. For activation, TCM was further supplemented with 1 µg/mL anti-CD3 (Bio X Cell) and 1 µg/mL anti-CD28 (Bio X Cell). After overnight activation, T cells were transduced by spin infection with retroviral particles with 10 µg/mL polybrene (Sigma). Spin infection was performed by centrifugation at 850g for 1.5 h at 32°C. T cells were kept in TCM supplemented with 100 U/mL human IL-2 (Peprotech) for 4–6 d prior to analysis for CD4 expression.