RNA sequencing of isogenic BRCA2 haploinsufficient vs. wild-type T-ALL cells

We found a high frequency of heterozygous Fanconi-BRCA pathway mutations in pediatric T-ALL. BRCA2 was the most commonly mutated gene. We transduced Cas9-expressing Jurkat cells, which lacked an identifiable BRCA2 mutation, with an integration-defective lentiviral guide RNA expression construct targeting exon 11 of BRCA2 (NM_000059). Single-cell cloning and sequencing analysis revealed two distinct clones harboring monoallelic BRCA2 frameshift mutations, termed clones W4 and W5. Each of these clones was subjected to RNA sequencing analysis. RNA sequencing was performed on 2 independent BRCA2 haploinsufficient Jurkat clones and 2 controls (parental and Cas9-transduced) Jurkat clones, using paired end 75 bp reads on an Illumina NextSeq 500 instrument.