GSM6067792: MEF1; Mus musculus; RNA-Seq
RNA was extracted and purified from young and aged HSC (20,000-25,000 cells from individual mice) using Direct-zol RNA micro kit (Zymo Resarch) according to the manufacturer's instructions (RNA integrity number value >8) 3-prime biased approach was used to generate sequencing libraries. An 8bp sample index anda. 10bp unique molecular identifiery (UMI) were added during the initial poly(A) priming and pooled samples were amplified using a template swithcing oligonucleotide.  The Illumina P5 (5' AAT GAT ACG GCG ACC ACC GA 3') and P7 (5' CAA GCA GAA GAC GGC ATA CGA GAT 3') sequences were added by PCR and Nextera transposase, respectively. The library was designed so that the forward read (R1) utilizes a custom primer (5' GCC TGT CCG CGG AAG CAG TGG TAT CAA CGC AGA GTA C 3') to sequence directly into the index and then the 10 bp UMI. The reverse read (R2) uses the standard R2 primer to sequence the cDNA in the sense direction for transcript identification.