Subcellular fractionation for HEK293 cells was performed as previously described31 (link) with the following modifications. Cell pellets were first resuspended in 200 µL cold cytoplasmic lysis buffer using wide orifice tips and incubated on ice for 6 minutes. The subsequent steps were as described previously31 (link), with the exception that for each collected subcellular fraction, 5% was used for immunoblot analysis, and then 1 mL Trizol LS (Life technologies) was added to the remaining aliquot for RNA purification using the QIAGEN RNA cleanup protocol.
Fractionation for mES cells was carried out as previously described30 (link). We confirmed by imaging the efficacy of fractionation using DAPI to stain for intact nuclei, and used ER-tracker red (BODIPY TR Glibenclamide, Thermo Fischer Scientific) to confirm removal of ER contaminants in the nuclear fractionations. Ribolock RNase inhibitor (Thermo Fischer Scientific) was used to prevent RNA degradation.
Fractionation for mES cells was carried out as previously described30 (link). We confirmed by imaging the efficacy of fractionation using DAPI to stain for intact nuclei, and used ER-tracker red (BODIPY TR Glibenclamide, Thermo Fischer Scientific) to confirm removal of ER contaminants in the nuclear fractionations. Ribolock RNase inhibitor (Thermo Fischer Scientific) was used to prevent RNA degradation.
