GSM8711812: Pooled CD45.1+ Cells, Nov 06, Lane002; Mus musculus; RNA-Seq
Bone marrow cells from mice receiving treatments were washed twice with cold PBS before labeling with unique TotalSeqA-compatible HashTags. Subsequently, samples were incubated with mouse Fc receptor-blocking agent and labeled with DAPI for live cell sorting by flow cytometry using an Aria cytometer. After sorting, equal numbers of cells were pooled and submitted for sequencing. A 10X Genomics Chromium machine was used for 3000-5000 single-cell capture. Cells were washed and partitioned into nanoliter-scale Gel Bead-In-Emulsions (GEMs), where all generated cDNA share a common 10x Genomics Barcode but uses a pool of ~750,000 barcodes to index each cell's transcriptome. The silane magnetic beads and Solid Phase Reversible Immobilization beads were used to clean up the GEM reaction mixture and the barcoded cDNA was then amplified in a PCR step. Libraries were prepared using the Chromium Single Cell 3' Reagent Kits (v3.1): Single Cell 3' Library & Gel Bead Kit v3.1, Single Cell 3' Chip G Kit v3.1 and Single Index Kit T Set A. The P7 and R2 primers were added during the GEM incubation and the P5 and R1 during library contruction via end repair, A-tailing, adapter ligation and PCR.