OEX002090
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OEX_Yuanliang_1911091431 |
Transcriptomic |
Total BM cells were harvested from primary leukemia and WT mice and subjected to RNA extraction with Trizol (Invitrogen). c-Kit+B220+MAC-1- (K+B+M-), c-Kit+B220+MAC-1+ (K+B+M++), c-Kit+B220+MAC-1++ (K+B+M+), and c-Kit+B220-MAC-1++ (K+B-M++) cells were sorted from SP of AML transplants and subjected to RNA extraction with RNeasy Plus Micro Kit (Qiagen, 74034).rRNA was removed using Ribo-Zero rRNA Removal Kits (Epicentre, MRZMB126). Subsequently, rRNA depleted samples were fragmented, then subjected to first and second strand cDNA synthetization. cDNAs were ligated with sequencing adapters according to standard Novaseq 6000 platform protocols.
Spleen (SP) cells were harvested from a Setd2-/- AML transplants mice, respectively. SP cells were stained with a biotin-c-Kit antibody for 15 minutes on ice and washed with MojoSort™ Buffer (BioLegend, 480017) separately. Cells were then labeled with Streptavidin Nanobeads (BioLegend, 480016) on ice and washed again 15 minutes later. Cell pellets were resuspended in 2.5 ml MojoSort™ Bufferbuffer and placed the tube in a magnet for 5 minutes, followed by collecting the magnetically labeled fraction twice. Subsequently, MACS isolated cells were stained with an antibody against c-Kit again, and sorted by BD FACSAria™ III (BD Biosciences). After purity, BD Rhapsody system was used to capture the transcriptomic information of single cells. The single cells were randomly sedimented in microwellssingle cells were randomly sedimented in microwells (>200,000) and captured by a limiting dilution approach. Beads with oligonucleotide barcodes were then added to reach a saturated state, ensuring that each cell can be paired with one bead. All beads were collected into a microcentrifuge tube. 6499 cells for WT and 7179 cells for leukemia were captured and used for scRNA-seq. Whole transcriptome libraries were constructed following the BD RhapsodyBD Resolve single-cell whole-transcriptome amplification workflow. Single-cell libraries were quantified using a High Sensitivity DNA chip (Agilent) on a Bioanalyzer 22100 and the Qubit High Sensitivity DNA assay kit (Thermo Fisher Scientific). The library for each sample was sequenced by HiSeq Xten (Illumina) on a 150 bp paired-end run.Libraries were loaded onto a Novaseq 6000 system and sequenced using a High Output sequencing kit (75× 2 bp) (Illumina). |
2019-11-09 |