GSM5965786: IRF2_KO_GEX; Mus musculus; RNA-Seq
For AB-Seq experiment, Tumors were cut into small fragments and digested in RPMI media containing collagenase I, FBS, DnaseI in a gentleMACS dissociator. Tumor ells were filtered and subjected to red blood cell lysis. CD45+ tumor immune cells were FACSorted and the sorted cells  were labelled with sample multi-plexing antibodies and AbSeq Ab-Oligos (antibody-oligonucleotides) using the BD Mouse Immune Single-Cell Multiplexing Kit. For ATAC-Seq experiment, Tumors were cut into small fragments and digested in RPMI media containing collagenase I, FBS, DnaseI in a gentleMACS dissociator. Tumor cells were filtered and subjected to red blood cell lysis. CD8+ tumor immune cells were isolated using autoMACS Pro Separator (Miltenyi Biotec), washed and treated with DNase I, and nuclei isolated according to the 10x Genomics nuclei isolation for single cell ATAC sequencing protocol. For CUT&Tag, naïve CD8+ T cells were isolated from the spleens and lymph nodes of C57BL/6 by negative selection (Stem Cell Technologies). Isolated CD8+ T cells were activated for 3 days using 5ug/mL plate-bound anti-CD3 (16-0031-86 Invitrogen) and 2ug/mL soluble anti-CD28 (16-0281-86 Invitrogen) antibodies in 10% RPMI complete media containing 100U/ml rhIL2 (200-02 PeproTech). Nuclei were isolated and CUT&Tag performed using the protocol from Epicypher (EpiCypher® CUTANA? Direct-to-PCR CUT&Tag Protocol v1.6 Revised: 11.04.2021). Prior to sequencing, samples were normalized based on their DNA concentrations and pooled. For AB-Seq experiment, Whole transcriptome Analysis (WTA), AbSeq and Sample Tag libraries were constructed using the BD Rhapsody? WTA Amplification Kit from cDNA encoded on beads after cell capture on the BD Rhapsody? Single-Cell Analysis System, using the manufacturer's recommended protocol.. For ATAC-Seq experiment, ATAC libraries were prepared following the 10x Genomics sequencing workflow and protocol. For CUT&Tag library preparetion was performed using the protocol from Epicypher (EpiCypher® CUTANA? Direct-to-PCR CUT&Tag Protocol v1.6 Revised: 11.04.2021).