GSM4294423: BDL day 14 [KW101218-11_S11]; Mus musculus; RNA-Seq
Livers were harvested, then minced and digested with PBS containing 0.1% collagenase (Sigma), 0.1% pronase (Sigma), and 0.01% DNase (Sigma) for 45min at 37oC. After addition of FBS (10% final concentration), the dissociated cells were strained through a 70 um pore filter and red blood cells were removed with RBC lysis buffer. The resultant cells then underwent serial centrifugation to enrich the nonparenchymal fraction. Cells were incubated with propidium iodide and were analyzed by FACS Aria I (BD) at the CHLA FACS Core. After exclusion of dead cells stained with propidium iodide, expression of GFP were analyzed. Negative gating was set with simultaneously digested wild type livers. For RNA-seq analysis, we sorted 1x10e5 GFP(+) cells directly into Trizol solution. Total RNA were purified using Trizol solution. RNA were further purified with silica bead Microprep columns (Qiagen, Germantown, MD). RNA quality was analyzed with an RNA Bioanalyzer (Agilent, Santa Clara, CA). Libraries were prepared with a Truseq mRNA library prep and sequencing was performed on an Illumina Nextseq Mid-Output flowcell (Illumina, San Diego, CA) at the CHLA sequencing core.
GEO Accession
GSM4294423