GSM7663634: FACS-sorted cKIT(+) spermatogonia, Cbx2 amplicon, Mut band, Rosa26-CreERT2; Cbx2[Flox/Flox], rep4; Mus musculus; RNA-Seq
c-KIT(+) spermatogonia were FACS-sorted to concentration of  about 1000 cells/µL, and a total ~8000 cells were loaded into the Chromium controller. We used 5' barcoding instead of 3' barcoding because deleted exons of Cbx2 were close to the 5' end of the gene. Single cell analysis with targeted genotyping was done by adapting 'Genotyping of Transcriptome (GoT)' (Nam et al., 2019, PMID: 31270458) and V(D)J region amplification of T or B-cell receptor protocol by the 10X Genomics. We aimed to make specific Cbx2 amplicons from cell-barcoded cDNA to amplify cDNA fragments and sequence Exon5 junctions to distinguish wild type (Exon4 spliced to Exon5) and mutant (Exon2 spliced to Exon5 after the deletion of Exon3, 4) for specific cell barcodes. Cbx2 inducible knock out & FACS-sorted cKIT(+) spermatogonia profiling : single cell barcoded cDNA was made with Chromium Next GEM Single Cell 5' Reagent Kits (10X Genomics, Cat#:1000265) based on the manufacturer's protocol with 14 PCR cycles of cDNA amplification. 50 ng of the amplified cDNA based on the manufacturer's protocol. 14 cycles of library amplification indexing PCR was performed. To generate Cbx2 amplicons, we performed nested PCRs to amplify Cbx2-specific cDNAs with cell barcodes. In the second PCR step of the nested PCR, Cbx2-specific primer that has a partial Illumina read2 sequence was used. The Cbx2-specific second nested primer also contains 'stagger' sequences between the Cbx2-specific region and partial read2 to increase library complexity for accurate base calls during Illumina sequencing. We confirmed the amplification of the correct-sized bands corresponding to wild-type and deleted Cbx2 amplicons by agarose gel electrophoresis. The amplified bands were excised and gel purified. Index PCR was performed to make the final Illumina-compatible libraries with unique indices with 10X provided index-PCR primers. Cbx2 amplicon libraries were pooled with standard 10X single cell gene expression libraries to occupy ~2% of total reads. A total of 4 samples were sequenced in one P3 lane on a NextSeq 2000 (NextSeq 2000 P3 Reagents (100 Cycles) Cat:20040559).