GSM6828967: P12#2; Mus musculus; RNA-Seq
Treated mice where euthanized, femur and tibia extracted, crashed in sample medium (PBSx1, EDTA 2mM, serum 2%) and loaded on Histopaq1083. Antibodies of Biolegend: CD45.1 APC, CD45.2 pacific blue, Mac1 PE-Cy7, B220 APC-Cy7, CD3e PE, Lineage-PacificBlue (Including- anti Ter119, Mac1, Gr1, CD3e, CD4, CD8 and B220), c-Kit Alexa780 , Sca1- APC , CD150-PEcy7 , CD48- percp cy5.5 , CD9-APC, CD34-PE, CD84-PE. Cells were stained on ice for 1 hr, and washed. Flow cytometry analy-sis of the reporter expression frequencies (ZsGreen+), and the surface markers expression, was performed on the Gallios Flow Cytometer (Beckman Coulter). FACS data analysis was performed using Kaluza v1.2 (Beckman Coulter). Sorting was performed on FACSAr-ia III (BD), essentially as previously reported [Keinan et al; Cell Death & Disease (2021) 12: 193]. 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.