GSM6031763: TMRM_Low3; Mus musculus; RNA-Seq
In brief, intact bone marrows from mice at the age of 6-8 weeks 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 Lin– Scal1+ c-Kit+ CD48– CD150+ (HSC) (3-5 µL) of each sample were transferred into 40 µL cell lysis buffer prepared by adding 2 µL of RNase inhibitor to 38 µL of a 0.25% (v/v) 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 of each sample. cDNA purified with VAHTS DNA Clean Beads (N411, Vazyme) was used to perform PCR enrichment with 13 cycles of amplification. Enriched cDNA was quantified using Qubit dsDNA HS Assay Kit (Q3285, Guangzhou IGE biotechnology ltd) after purification. TruePrepTM DNA library Prep Kit V2 (TD503, Vazyme) was used to prepare RNA seq library following the manufacturer's protocol. Libraries were sequenced as paired 150 bp using NovaSeq-PE150.