title: GSM2402668: CMP_M33_Het_8; Mus musculus; RNA-Seq
library_construction_protocol: Liver single cell suspensions by homogenization of fetal liver tissue with a 1ml Eppendorf pipette and Hank ́s buffered salt solution (HBSS) with 3% foetal calf serum (FCS) were prepared. Cells were then stained with anti-CD45.2-FITC (BD Pharmingen), biotinylated lineage antibodies (anti-B220, -CD3,Gr-1, and -Ter-119; all BD), anti-Mac1/CD11b-eFluor 450 (eBioscience), anti-CD117/c-kit-Alexa Fluor®780 (eBioscience) and anti-Sca-1-PeCy7 (eBioscience). Biotinylated monoclonal antibodies were labeled by incubation with Streptavidin-PerCP/PerCP-Cy5.5 (eBioscience) and cells sorted directly into lysis buffer (0.2%TritonX supplemented with RNAse Inhibitor (Promega)) RNA was cleaned-up from the crude lysate with Agencourt RNAclean XP SPRI beads (Beckman-Coulter). cDNA was synthesized and pre-amplified from 5 μ l of lysate as described in Picelli et al 2014 (Smart-seq2). 0.7 ng of pre-amplified cDNA was used as input for tagmentation by the Nextera XT Sample Preparation Kit (Illumina), where a second amplification round was performed for 12 cycles. For each sample, 5 ng of final library was pooled.
identifiers.namespace: GEO
identifiers.id: GSM2402668