GSM1315845: Fib-4D; Mus musculus; RNA-Seq
For fibroblasts: RNA was harvested using Trizol reagent.For hematopoietic cells: each of the sample, 25 individual cells were manually picked and transferred into lysis buffer by a mouth pipette, then directly reverse transcribed the released mRNA by a polyT primer, after that, polyA was added to the 3’ end of the first cDNA strand by terminal deoxynucleotidyl transferase, and the second strand cDNA was synthesized by another polyT primer, which was followed by 20+9 cycles of PCR to amplify cDNAs. For fibroblasts: RNA libraries were prepared for sequencing using standard Illumina protocols. Basically the mRNA was first purified and fragmented. First and second strand cDNAs were synthesized and end repaired, then 3’ end of the cDNA was adenylated and cDNAs were ligated to adaptors. After purification, the adaptor ligated cDNAs were enriched by primers that annealed to the adaptors. The library were validated on an Agilent Technologies 2100 Bioanalyzer.For hematopoietic cells: RNA-seq library was prepared following protocol of NEBNext Singleplex (#E7350) Oligos for Illumina. The cDNAs were fragmented to 200-400 bp by ultrasonication, the fragmented cDNAs were then end repaired, and cleaned up using AMPure XP® Beads (Beckman Coulter , Inc.). dA-tail were added to the end repaired cDNA, and NEBNext Adaptor was then ligated to the dA-tailed cDNA. The adaptor ligated cDNAs were PCR enriched with Universal PCR primer and Index primer, and the enriched cDNAs were then purified by AMPure XP® Beads. The library was quality checked on a Bioanalyzer ® (Agilent Technologies, Inc.).