Homozygous EGFP-knocked-in at the N terminus of BLIMP1 (EGFP-BLIMP1)
cell sorting
FACS (GFP+, EPCAM+)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Intestinal epithelium were isolated as described (Gracz et al., 2012) with minor modifications. The intestine (E16.5) 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. 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 and EpCAM positive cells were purified with FACS. Chromatin immunoprecipitation was performed as described previously(Kurimoto et al., 2015). Libraries for ChIP_seq were prepared as described (Kurimoto, 2015 Cell Stem Cell) with a modification for application to the Illumina systems. Briefly, ChIP-ed and input DNAs were sheared to an average size of 150 bp by ultra-sonication (Covaris S2). The sheared DNAs were end-repaired, dA-tailed, ligated to an amplification adaptor (P1-T Adaptor/F and Barcode-Internal+12-mer/R), amplified with a 10-cycle PCR using Library PCR primer 1 (Life technologies) and Library PCR primer Barcode001 + Internal adaptor. For sequencing on the Illumina systems, we then added an adaptor and index to the amplified library with two rounds of additional PCRs. The 1st round additional PCR was performed for 6 cycles with Read1-P1 and Read2-internal-adaptor primers. The 2nd round PCR was performed for 4 cycles with Illumina P5-Read1 primer and P7-index N-Read2 primer to produce the index-tagged library DNAs. To exclude the initial constant region consisted of the amplification adaptor sequence (P1-T Adaptor) and maximize the efficient sequencing reads, we designed a custom sequencing primer, Custom-primer-29mer (Supplementary Table S2 of the manuscript), which met sequencing on Illumina platform (Caporaso, 2011 PNAS) in terms of the length, GC content, and melting temperature. The DNAs were then sequenced on the HiSeq 2500 platform (Illumina) in high throughput mode to generate single-end 100-bp reads, as the manufacturer's instruction.