title: GSM5577958: Day 0 non-infected Lin– c-Kit+ bone marrow; Mus musculus; RNA-Seq
library_construction_protocol: Lineage marker negative cells (Lin−) were isolated from bone marrow or spleen using a MACS lineage cell depletion kit from Miltenyi Biotec. c-Kit+ and c-Kit− cells were separated by MACS using a CD117 MicroBeads kit from Miltenyi Biotec. Purity was assessed by labeling cells with the non-competing anti-c-Kit antibody (CD117; clone 3C11) from Miltenyi Biotec. Where indicated, purified cells were labelled with CFSE (Invitrogen). For obtaining HSPC-derived macrophages, purified Lin- cells were cultured in complete cell culture medium supplemented with 20 ng/ml Stem Cell Factor (SCF, Preprotech) and 50 ng/ml M-CSF (Preprotech) for 7 days. Single-cell GEMs were generated using a Chromium Controller instrument (10x Genomics). Sequencing libraries were prepared using Chromium Single Cell 3' V3 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. 10X Genomics Chromium 3' V3 chemistry. Libraries were sequenced on an Illumina HiSeq 4000 with 100 bp paired-end reads.