GSM9798408: Genome-wide BRIE screen day 5 chronic stimulation Prdm1-high bin; Mus musculus; OTHER
Genomic DNA was isolated from sorted cell populations, input cell populations, tumor-infiltrating T cell samples, draining-lymph-node samples, or plasmid-library controls for guide-count sequencing. Standard cell pellets were processed using QIAamp DNA Mini Kit or QIAamp Mini spin-column workflows. Fixed or low-input sorted material was recovered using SPRI-based DNA cleanup where appropriate. Tumor-infiltrating T cell guide-count samples were prepared from tumor digests enriched over Lympholyte-M and/or with CD8 TIL MicroBeads before pelleting or freezing for genomic DNA isolation. The extracted genomic DNA or plasmid DNA was used as input for integrated sgRNA cassette amplification. Integrated sgRNA cassettes were amplified from genomic DNA using indexed Illumina-compatible primers following Broad/GPP pooled-screen PCR workflows. Standard pXPR guide-count PCR used up to 10 ug genomic DNA per 100 uL reaction with Titanium Taq DNA polymerase, DMSO, a staggered P5 primer mix, and a unique indexed P7 primer. Primer sets were selected for the vector backbone, including pXPR_045-compatible and pXPR_206 or pRDA_206-derived vectors. For lower-input or nested tissue workflows, PCR cycle number was selected by qPCR using Q5 Hot Start high-fidelity polymerase, and available genomic DNA was split across multiple reactions to limit PCR jackpotting. No-template controls and test gels were used to check contamination and expected amplicon size. PCR products were pooled by sample and purified by AMPure XP cleanup, double-sided AMPure XP size selection at 0.6x/1.2x, or gel extraction depending on amplicon workflow and input complexity. Purified amplicons were quantified by Qubit and Bioanalyzer or TapeStation, pooled, and sequenced on Illumina MiSeq or NextSeq instruments using custom sequencing primers where required.
GEO
GSM9798408_r1