GSM2492109: zebrafish1 FAIRE-seq; Danio rerio; FAIRE-seq
To isolate zebrafish IECs, intestines were dissected, splayed, and washed extensively with ice-cold 1x PBS with care taken to remove as much intestine associated fascia, adipocytes, and blood vessels as possible. Three washed intestines were transferred into dissociation reagent 1 (DR1; 30 mM EDTA, 1.5 mM DTT, 0.5x Complete protease inhibitors (Roche), in 1x PBS) for 15 minutes on ice. Segments were transferred to Dissociation Reagent 2 (DR2; 30 mM EDTA, 0.5x Complete protease inhibitors (Roche), in PBS) and moderately shaken by hand for 5 minutes until most epithelial cells were isolated in the suspension. Intestinal lamina propria was removed and 8 ml of cold 1x PBS was added to the cells on ice. Cells were pelleted at 500 x G at 4°C, washed once with 13 ml of cold 1X PBS, and re-suspended in 0.5 ml cold 1x PBS. A 0.4 ml fraction was used for FAIRE, and 0.1 ml fraction was reserved for RNA extraction. Isolated IECs were placed in TRIzol Reagent for RNA extraction. For FAIRE, freshly isolated intestinal epithelial cells were directly fixed for 5-10 minutes in 10 ml of 1-3% w/v Formaldehyde solution (in 1x PBS) at room temperature and gentle rocking. Glycine (2.5M) was added to a final concentration of 125 mM to quench the formaldehyde. Cells were pelleted at 600 x G and washed three times in cold 1x PBS without dissociating the pellet. Fixed and washed cell pellets were flash frozen and stored at -80°C. Cells were lysed in 2 ml Lysis Buffer A (10 mMTris-HCl (pH8.0), 2% (vol/vol) Triton X-100, 1% SDS, 100 mM NaCl and 1 mM EDTA) and sonicated using a Branson Sonifier 450D equipped with a microtip for 6-13 cycles (1 second burst, 0.5 second pause, for 30 seconds/cycle at 70% intensity) allowing samples to cool on ice for 1 minute between cycles. Isolated IECs were placed in TRIzol Reagent. UNC Highthrough-put Sequencing Core performed TruSeq library preperation.