GSM9723621: single-cell RNA sequencing of combined CD45+ TBM, LK and LSK compartment from Idh1-WT bone marrow, rep 2; Mus musculus; RNA-Seq
To isolate live CD45+ total hematopoietic cells, 5*106 cells per bone marrow were stained with CD45.1 and DAPI. Remaining total bone marrow cells were subjected to lineage depletion as described above. Upon lineage depletion, cells were stained with fluorophore coupled antibodies. All cell stainings were performed for 30 min at 4°C on a rolling platform. Upon incubation, cells were washed with 30 mL PBS/2%FBS and were resuspended in 300 µL PBS/2%FBS and directly subjected to fluorophore activated cell sorting at BD FACS Aria II cell sorter. Per mouse, 4000 cells were sorted from the following populations: total hematopoietic cells defined as EYFP+DAPI-CD45.1+, LSK defined as EYFP+DAPI-Lin-Sca1+cKit+ and LK defined as EYFP+DAPI-Lin-cKit+. Per mouse, all cell populations were sorted into a single tube containing 300 µL StemSpan. Upon cell sorting, each 1.5 mL tube was filled up with fresh PBS/2%FBS and tubes were kept on ice until library preparation. Library preparation was performed at the “single-cell Open Lab” (scOpenLab) at the DKFZ by following manufacturer's instruction using the Single Cell 3' Reagent Kits v2 from 10X Genomics.  Directly after cell sorting, cells were pelleted at 300 g for 5 minutes and completely subjected to reverse transcription. For that, cells from each mouse, reverse transcription (RT)-master mix, partitioning oil and gel beads were loaded onto a “Chip A Single Cell” where “Gel Bead-In-Emulsions” (GEMs) were produced using a Chromium Controller (10x Genomics). This step was performed by Katharina Bauer. Afterwards, GEMs were transferred into PCR tube strip and reverse transcription was performed in a thermal cycle followed by cDNA clean-up and cDNA amplification. Amplified cDNA was cleaned up and library construction was performed by fragmentation, end repair and A-tailing followed by adapter ligation and library amplification and indexing PCR. Libraries were sequenced on a NovaSeq 6000 S4 (Illumina) in a paired-end (PE 26/96 bp) mode at the GPCF at DKFZ. Library preparation was performed by me and was supervised by Dr. Mark Hartmann.