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Blue:RNA-Seq Experiments:11102016
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==Nuclei isolation protocol used== Nuclei isolation from Cells #Prepare cells: ##Wash with PBS and centrifuge 300g 5min ##Resuspend cells to 1x106 per ml in PBS #Dounce with sucrose-buffered extraction buffer (RNAse-free) ##Wash dounce in RNaseAway or RNaseZap, rinse with Milli-Q water ##Add 1ml of NEB containing 1ul DAPI (5mg/ml) to dounce and chill on ice ##Add 250ul cells to 1 mL ice cold NEB buffer - incubate 10 minutes on ice ##Gently dounce sample on ice: ###5 strokes with loose pestle A ###~10 strokes with pestle B (minimize bubble formation) ##Pass debris through 30-Β΅m CellTrics filter to 15-mL conical tube ##Wash dounce with 1 mL NEB buffer, and filter this into the same tube ##Bring up to 10 ml with PBSE ##Pellet nuclei: 900g, 10 min. ##Resuspend pellet in 1ml PBSE+1%BSA. Add 9ml PBSE. ##Pellet nuclei: 900g, 10 min. ##Resuspend pellet in 1ml PBSE+1%BSA.
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