title: GSM4725223: EG_SERIES_3: PROG_MTB; Mus musculus; RNA-Seq
library_construction_protocol: Bone marrow cells were harvested from femurs, tibiae, humeri and ilia, and incubated with the described biotin-conjugated antibodies against lineage-committed cells. Subsequently, the cells were incubated with MACS streptavidin conjugated microbeads (Miltenyi) for 30 minutes at 4°C. A depletion of the lineage-committed cells was performed using AutoMACS (Miltenyi) to reduce the contaminating total cell number. The remaining cells were incubated with the corresponding antibodies for LKS+ and cKit+ progenitors Single-cell GEMs were generated using a Chromium Controller instrument (10x Genomics). Sequencing libraries were prepared using Chromium Single Cell 3' Reagent Kits (10x Genomics), according to the manufacturer's instructions. Briefly, GEM-RT was performed in a thermal cycler: 53°C for 45 min, 85°C for 5 min. cDNA was cleaned up with DynaBeads MyOne Silane Beads (ThermoFisher Scientific) and amplified with a thermal cycler: 98°C for 3 min, cycled 12 x 98°C for 15 s, 67°C for 20s, 72°C for 1 min, and 72°C 1 min. After a cleanup with SPRIselect Reagent Kit, the libraries were constructed by performing the following steps: fragmentation, end-repair, A-tailing, SPRIselect cleanup, adaptor ligation, SPRIselect cleanup, sample index PCR, and SPRIselect size selection. Libraries were sequenced on a NovaSeq S2 flowcell with 100 bp paired-end reads