title: GSM3904792: PBS-3 A3_3_S18; Mus musculus; RNA-Seq
library_construction_protocol: Treated mice where euthanized, femur and tibia extracted, crashed in sample medium (PBSx1, EDTA 2mM, serum 2%) and loaded on Histopaq1083. this mononuclear enriched population was stained and sorted for Lineage-Sca1+cKit+CD150+Fgd5-mCherry+ cell, 100 cells from each sample, to 384 plate with lysis buffer also containing RNasin that blocks RNA degradation and stabilizes the RNA. library construction and sequencing was performed by the INCPM facility (Rehovot). in short - library preparation was done according to the Smart-seq2 protocol (Simone Picelli et al. Nature Protocols 2014) samples were pooled together and sequenced on one lane of the illumina HiSeq 2500 V4 instrument. SMARTSeq-2 Ultra Low Input RNA V4 kit from Clontech (polyA based capture), with 14 cycles of cDNA amplification. The amplified cDNA was fragmented using the Covaris machine to around 450bp. Illumina libraries were constructed using the End repair, A base addition and adapter ligation (TruSeq like adapters:GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTCGCCTTATCTCGTATGCCGTCTTCTG) and amplified using 13 PCR cycles. Performed by INCPM in the Weizmann institute of science.
attributes.tag: GEO Accession
attributes.value: GSM3904792