P4 pancreas islets -2
Mus musculus
Protocols: Islets were dispersed to a single cell suspension using TrypLE Express (Invitrogen). Cells were passed through a 40 um filter (BD Falcon 352340) and washed with PBS at least twice. Cells were fixed and permeabilized with 4% PFA (Electron Microscopy Sciences), 0.1% saponin (Sigma-Aldrich 47036) in molecular grade PBS (Ambion) supplemented with 1:100 RNasin Plus RNase Inhibitor (Promega, N2615) for 30min at 4C. All the subsequent steps were carried out at 4C. Cells were pelleted by centrifugation at 3000g for 3min at 4C and washed in Wash Buffer: PBS containing 0.2% BSA (Gemini Bio-Products), 0.1% saponin (Sigma-Aldrich), 1:100 RNasin Plus RNase Inihibitor. Primary antibody staining was carried out while 3D rocking for 30min at 4C in Staining buffer containing PBS with 1% BSA, 0.1% saponin and 1:25 RNasin Plus RNase Inhibitor (note that certain antibodies may require longer incubation times). Cells were washed twice in Wash Buffer followed by secondary antibody staining in Staining buffer. Following secondary antibody staining cells were washed twice in Wash buffer and resuspended in Sort buffer containing PBS, 0.5% BSA, and 1:25 RNasin Plus RNase Inhibitor. Fixation, washing, staining and sorting were performed at a concentration of 5-10 M cells/ml. (Hrvatin et al., PLoS ONE 2014) After sorting, cells were pelleted by centrifugation at 3000 g for 5min at 4C. The supernatant was discarded. Total RNA was isolated from the pellet using the RecoverAll Total Nucleic Acid Isolation kit (Ambion), starting at the protease digestion stage of manufacturer-recommended protocol. The following modification to the isolation procedure was made: instead of incubating cells in digestion buffer for 15 minutes at 50C and 15 minutes at 80C, we carried out the incubation for 3 hours at 50C. Cell lysates were frozen at -80C overnight before continuing the RNA isolation by the manufacturer's instructions. NA
ENA-FIRST-PUBLIC
2015-03-17T17:02:55Z
ENA-LAST-UPDATE
2018-03-08T23:31:13Z
External Id
SAMEA3307909
INSDC center name
Massachusetts Institute of Technology
INSDC first public
2015-03-17T17:02:55Z
INSDC last update
2018-03-08T23:31:13Z
INSDC status
public
Submitter Id
E-MTAB-3418:P4 pancreas islets -2
age
P4
broker name
ArrayExpress
common name
house mouse
sample name
E-MTAB-3418:P4 pancreas islets -2
scientific_name
Mus musculus
strain
Swiss-Webster