title: GSM2420083: P14_intestinal epithelium RNA-Seq, rep1; Mus musculus; RNA-Seq
library_construction_protocol: Intestinal epithelium were isolated as described (Gracz et al., 2012) with minor modifications.  The intestine (P14) from caudal half of duodenum to the ileocecal junction of EGFP-BLIMP1 mice were dissected, and the surrounding connective tissues and vessels were gently removed. Then the intestine was open by cutting longitudinally through the lumen The isolated intestine were placed in the 1.5 ml eppendorf tube containing ice-cold PBS and cut into small pieces as fine as possible.  The intestine fragments were then transferred to 10 ml Falcon tube containing dissociation reagent ♯1 [47 mL PBS, 3 mL of 0.5 M (30 mM) EDTA, 75 μL of 1 M (1.5 mM) DTT] and placed on ice for 20min.  After the incubation, dissociation reagent ♯1 was replaced with dissociation reagent ♯2 [47 mL PBS, 3 mL of 0.5 M (30 mM) EDTA].  After 10 min incubation at 37 °C, the tube that contained intestine was shaken by hand for 30 sec to release epithelium from the basement membrane.  Dissociated cells were washed by removing supernatant and re-suspending cells in 10 mL PBS with 10% FBS.  The cells were collected by centrifugation and then re-suspended in 10 mL DMEM containing 8-mg Dispase (GIBCO), which digested cell–cell junction proteins and dissociated the whole epithelium to single cells.  The cells were then incubated in a water bath for 10 min at 37°C with vigorous shaking every 2 min.  After adding 10 % FBS, the cell suspension was passed through the cell strainer to remove large undissociated clumps of cells and connective tissues. BLIMP1 negative and EpCAM positive cells were purified with FACS. Total RNAs from collected cells were extracted with RNeasy Micro Kit (QIAGEN, 74004) according to the manufacture's instruction. RNA-seq libraries were prepared according to Nakamura et al., 2015, GEO:  GSE63266.
attributes.tag: GEO Accession
attributes.value: GSM2420083