title: P13 pancreas islets -2
organism: Mus musculus
description: 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
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2015-03-17T17:02:55Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T23:33:46Z
attributes.tag: External Id
attributes.value: SAMEA3307913
attributes.tag: INSDC center name
attributes.value: Massachusetts Institute of Technology
attributes.tag: INSDC first public
attributes.value: 2015-03-17T17:02:55Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T23:33:46Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-3418:P13 pancreas islets -2
attributes.tag: age
attributes.value: P13
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: sample name
attributes.value: E-MTAB-3418:P13 pancreas islets -2
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: strain
attributes.value: Swiss-Webster