GSM5077721: Control NG2 Rep1; Mus musculus; RNA-Seq
Spinal cord tissues were collected from perfused female mice as previously described (PMID: 32284594). Single-cell suspensions were prepared from entire spinal cords following the adult brain dissociation (ABD) kit manufacturer's instructions with modification (Miltenyi Biotec). Briefly, minced tissues were individually incubated with ABD Mix 1 containing 15 μM actinomycin D (ActD; Sigma) (PMID: 29024657) for 15 min at 34°C, and then ABD Mix 2 was added to solution for 10 min at 34°C. Tissues were gently triturated and then incubated for 10 min at 34°C. Homogenized tissue solutions were passed through 70-μm smartstrainer (Miltenyi Biotec), washed with cold Dulbecco's phosphate-buffered saline and centrifuged at 450xg for 7 min at 4°C. Tissue debris was removed following the ABD Kit debris removal step instructions and then passed through 30-μm smartstrainer (Miltenyi Biotec) and centrifuged at 450xg for 7 min at 4°C. All steps above were performed in the presence of 3 μM ActD. Single-cell suspensions were incubated with 1 μM Sytox blue live/dead stain (Thermo Fisher Scientific) for 5 min at 4°C and then cell sorting was performed on an FACSARIA II (BD Biosciences). Sytox blue– NG2-tdtomato+ cells were sorted directly into tubes containing RLT plus lysis buffer (Qiagen) supplemented with 1% 2-mercaptoenthanol (Sigma) and 0.25% reagent DX (Qiagen). Cell lysates were frozen on dry ice and immediately stored at -80°C until use Frozen NG2 cell lysates in RLT buffer were thawed at 24°C and then lysed using the QIAshredder (Qiagen) following manufacturer's instructions. Total RNA was isolated from cell lysates using the RNAeasy micro kit without modification (Qiagen). RNA quality and quantity were determined by Bioanalyzer pico chip analysis (Agilent) and all samples with RNA integrity number > 8 were used for RNA-seq library preparation. cDNA libraries were generated from total RNA using the Ovation RNA-seq System V2 (NuGEN). Libraries were quantified and quality checked by KAPA qPCR (Roche) and Bioanalyzer DNA chip analysis (Agilent), respectively. Libraries were pooled and paired-end 75 base pair read length sequenced across 8 lanes on a Nextseq500 (Illumnia), for a sequencing depth of >40 million reads per library. FASTQ files were generated in Biospace following manufacturer's guidelines (Illumina).
GEO
GSM5077721