title: GSM5145635: SCD1_CMo; Homo sapiens; RNA-Seq
library_construction_protocol: Total monocytes from patients with SCD and HD were enriched from prepared PBMC using a pan monocyte isolation kit (Miltenyi Biotec, Auburn, CA) following manufacturer's instructions. After that enriched cells were further purified with flow cytometer sorter. Cells were stained with fluorescently labeled anti-human CD14, CD16, HLA-DR and CD45 antibodies, and DAPI. Single DAPI-CD45+HLA-DR+CD14+CD16- classical monocytes were sorted using FACSAria Cell Sorter (BD Biosciences). Sorted cells were lysed and subjected to mRNA isolation with the Dynabeads mRNA Direct Purification kit (Thermo Scientific). We use Agilent 2100 Bio analyzer (Agilent RNA 6000 Nano Kit) to do the total RNA sample QC: RNA concentration, RIN value,28S/18S and the fragment length distribution. 1)mRNA enrichment: Oligo dT Selection or rRNA depletion; 2) RNA fragment and reverse transcription: Fragment the RNA and reverse transcription to double-strand cDNA (dscDNA) by N6 random primer; 3) End repair, add A tailing and adaptor ligation: The synthesized cDNA was subjected to end-repair and then was 3' adenylated. Adaptors were ligated to the ends of these 3' adenylated cDNA fragments; 4) PCR amplification: The ligation products were purified, and many rounds of PCR amplification were performed to enrich the purified cDNA template using PCR primer;5) Denature and cyclization: Denature the PCR product by heat and the single strand DNA is cyclized by splint oligo and DNA ligase.