GSM8740574: yHSC-2; Mus musculus; RNA-Seq
In brief, intact bone marrows from mice at the age of 2-4 months or 20-24 months were flushed from mouse femora and tibiae. Bone marrow cells by pipetting with 1 mL syringe, and then underwent red blood cell lysis using 0.16 M ammonium chloride solution. Low input RNA seq was performed according to published scRNA seq protocols with modifications (Picelli et al., 2014). Briefly, FACS-sorted 1500 HSCs in 3-5 μl PBS were transferred into 40 μl cell lysis buffer prepared by adding 2 μl of RNase inhibitor to 38 μl of a 0.25% (vol/vol) Triton X-100 solution. Quickly vortex the tube to mix, and then purify RNA using 80 μl VAHTS RNA Clean Beads (N412, Vayzme), following reverse transcription with 5 μl purified RNA and 15 μl RT mix. cDNAs were purified by VAHTS DNA Clean Beads (N411, Vazyme) for PCR enrichment with 12 cycles. Enriched cDNA was quantified using Qubit dsDNA HS Assay Kit after purification. TruePrepTM DNA library Prep Kit V2 (TD503, Vazyme) was used to prepare RNA seq library following the manufacturer's protocol. Individual libraries were sequenced as NovaSeq 6000 PE150 platform. At least three replicates were included.