GSM5053472: Lib_Lib28_sc72; Mus musculus; RNA-Seq
Bone marrow (BM) cells were flushed from the tibiae, femora, and pelvis. Spleen (SP) cells were ground by the mortar and pestle. BM or SP cells were then stained with anti-c-Kit microbeads (catalog no. 130-091-224, Miltenyi Biotech) and c-Kit+ cells were enriched by columns. These cells were stained with CD201 (eBio1560), CD150 (TC15-12F12.2), CD48 (HM48-1), Sca-1 (D7), c-Kit (2B8), and with a lineage-marker cocktail of B220 (RA3-6B2), Gr-1 (RB6-8C5), TER-119. Cells were sorted by FACSAria III cell sorter (Becton Dickinson, US) according to surface markers or GFP expression under the single-cell sorting mode. The scRNA-seq libraries were prepared based on a modified Smart-seq2(STRT-seq) protocol. Single cells were sorted into individual tubes of a 0.2 mL 8-strip PCR tube containing lysis buffer by FACS. In addition, the single-cell sorting mode was used for cell sorting with the appropriate side scatter pulse width (SSC-W) and side scatter pulse height (SSC-H) settings to avoid doublets. First-strand cDNAs were synthesized using 25-nucleotide oligo-dT barcoding primer and template switch oligo (TSO) primer. Following reverse transcription, the barcoded cDNAs were amplified via 20 cycles of PCR. Next, the cDNAs of each cell were pooled together and purified twice using 0.8 x Agencount AMPure XP beads. Biotin tag and indexing sequences were introduced by additional 4 cycles of PCR amplification using biotinylated primers. Purified once and sonicated (~300 bp by Covaris M220 system), then the 3' ends of the fragmented cDNA were enriched with Dynabeads Myone streptavidin C1 beads (Invitrogen). Finally, the cDNA libraries were generated using a Kapa Hyper Prep Kit (Kapa Biosystems) and sequenced on Illumina Hiseq X Ten (Novogene) using paired-end mode (2×150 bp). Modified Smart-seq2(STRT-seq)