GSM4859505: Monocyte mouse2; Mus musculus; OTHER
HSCs isolated from bone marrow from C57BL/6 mice were cultured ex vivo. Five days after plating, cultured HSCs were infected with barcoding lentivirus pBC008 in HSC culture medium. Two days after infection, medium was refreshed with HSC culture medium. Four days after infection, the infected HSCs were detached and GFP+LinSca1+cKit+ cells were sorted using a FACS Aria IIIa instrument and mixed with CD45.2+ LincKit+Sca1- cells as rescue cells. Five-week-old recipient CD45.2 male mice were irradiated with a dose of 9 Gy to the whole body and were intravenously injected with 2x10e4 GFP+LSK and 10e5 rescue cells per mouse. Two months later, peripheral blood, hearts, and kidneys were isolated. Then the sorted cells were lysed in buffer RLT plus (QIAGEN) and the genomic DNA was extracted from these samples by using AllPrep DNA/RNA Mini Kit (QIAGEN). Barcode sequences incorporated into genomic DNA were PCR amplified by NEBNext Ultra II Q5 Master Mix (NEB) with the following primers: 5'-TCT TTC CCT ACA CGA CGC TCT TCC GAT CTT AAC TTA CGG AGT CGC TCT ACG-3' and 5'-GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC TGG ATG GGA TTC TTT AGG TCC TG-3'. The cycling protocol was 98 °C, 30 sec; then 18 cycles of 98 °C, 10 sec, 60 °C, 10 sec, 72 °C, 30 sec; then 72 °C, 5 min. The PCR amplicons were amplified with NEBNext Multiplex Oligos (NEB) at 9-11 cycles to generate sequencing libraries.