Sample 10
Homo sapiens
Protocols: Tumor tissue was extracted from patient derived xenograft mice. RNA extraction (described below) was performed either on fresh tissue (control sample) or on cell populations after dissociation or dissociation followed by FACS sorting. For dissociation tissue was weighed and then minced using a scalpel or scissors to a size not exceeding 1 to 2 mm3. Tissue was then enzymatically dissociated into single cells using Horizon™ Dri Tumor & Tissue Dissociation Reagent (BD Biosciences, San Jose, CA) in a 37°C water bath for 30 minutes with frequent agitation. The enzymatic reaction was stopped by rinsing in PBS (Cellgro) and 1% BSA (Sigma-Aldrich). Dissociated tissue was filtered through a 70 mm sieve and treated with ACK Buffer (Life Technologies) to remove contaminating red blood cells. Cells were resuspended in PBS, without magnesium and calcium. FACS Sorting: Before staining, single-cell suspensions were blocked for 30 minutes, on ice in FcR block (Miltenyi Biotec). For surface marker characterization, cells were diluted in PBS and distributed into wells of a 96-well plate containing viability dye (LIVE/ DEAD Fixable Near-IR or Aqua, Invitrogen) and Hoechst 33342 (Invitrogen; at 0.2 mg/mL) for viable nucleated cell identification. Monoclonal antibodies used are listed in Table 2. Cells were incubated with antibody solutions for 30 minutes at 4C, in the dark, and then rinsed twice in PBS before acquisition on BD LSRII flow cytometer. For sorting, cells were stained using a four-color panel combining amine viability reagent, anti-mouse cocktail, and markers identified from the primary characterization plate. After staining, cells were rinsed, and resuspended in BD FACS Pre-Sort Buffer, filtered again through a 70 mm sieve, and populations of interest were sorted on the BD FACSAria II (BD Biosciences) at 20 p.s.i. using a 100- mm nozzle. Cell doublets or clumps were removed with electronic doublet discrimination gating. Post-sorting analysis of sorted subpopulations regularly showed purity >95%. Fresh tissue or cell populations were extracted using a Qiagen RNEasy kit according to the manufacturer's instructions. 20-50,000 cells stained for a specific surface marker were collected during FACS sorting and processed in bulk for RNA extraction. Quality assessments were determined using Agilent BioAnalyzer and NanoDrop readings. RNA specimens underwent QC analysis to determine their suitability for target labeling. QC analyses included determination of concentration and volume, A260/A280 ratio, and 28S/18S rRNA ratio (where applicable), and an RNA integrity summary score (Agilent RIN or Caliper RQS). RNA libraries were prepared for sequencing using Illumina RNASeq TruSeq Stranded mRNA SQ905 Kit.
ENA-FIRST-PUBLIC
2020-08-20T04:04:39Z
ENA-LAST-UPDATE
2020-08-10T16:58:04Z
External Id
SAMEA7171871
INSDC center name
Becton Dickinson Technologies
INSDC first public
2020-08-20T04:04:39Z
INSDC last update
2020-08-10T16:58:04Z
INSDC status
public
Submitter Id
E-MTAB-9428:Sample 10
broker name
ArrayExpress
cell type
Unsorted_3hr
common name
human
disease
Triple negative breast cancer
facs sorting
incubated 3h on ice
organism part
Patient derived xenograft
sample name
E-MTAB-9428:Sample 10
sample type
dissociated single cell suspension
scientific_name
Homo sapiens