Direct zol rna microprep kit

Samples were isolated for RNA according to the manufacturer's protocol using Direct-zol™ RNA MicroPrep kit (Genesee Scientific). RNA concentration was obtained by nanodrop, and cDNA was made using qscript cDNA supermix following the manufacturer’s instructions (Quantabio). cDNA was synthesized at 1000 ng using BioRAD iQ5 thermocycler. Cycles were: Priming for 5 min at 25 °C, RT: 30 min at 42 °C and RT inactivation for 5 min at 85 °C. cDNA was used to analyze gene expression by real time polymerase chain reaction (RT q-PCR) using PerfeCTa qPCR FastMixII (QUantbio). TaqMan assays used were Il6, Tnfα, Il1b, Gpnmb, Col1a1, Timp1, Mmp12, Rsad2, Ilrn1, Il1bp and 18S eukaryotic endogenous control (Thermo Fisher Scientific). Samples were normalized to 18S.

HFSCs from Rag1^–/– or Rag1^–/– CKO mice were sorted, spun down, and dissolved in TRIzol (Invitrogen, 10296028), and total RNA was harvested by means of the Direct-zol RNA MicroPrep Kit (Genesee Scientific, 11-330MB). Total RNA quality was determined with a spectrophotometer (Thermo Fisher Scientific, NanoDrop 2000). cDNA was then synthesized using iScript Reverse Transcription Supermix (Bio-Rad, 1708841), and qPCR was performed using SYBR Green Supermix (Bio-Rad, 1725275). Data were normalized to the expression of housekeeping GAPDH genes, and the relative abundance of transcripts was calculated by the 2^–ΔCT method. All primer sequences are listed in Table 2.
For RNA-Seq studies, HFSCs were sorted on day 20, day 26, and week 10 after the mice were born. For all samples, polyA⁺ RNA was isolated and RNA-Seq analysis was performed by BGI. Heatmaps, KEGG pathways, and GSEA were generated using Dr. Tom platform (https://biosys.bgi.com/). The RNA-Seq data sets were deposited in the NCBI’s Gene Expression Omnibus (GEO) database (GSE213603).