WT_ATAC_S518
Mus musculus
Protocols: CELL ISOLATION. We isolated small intestinal crypts from wild type (WT) (Villin-Cre -; Lsd1f/f) and intestinal-epithelial-specific knock-out (KO) (Villin-Cre+; Lsd1f/f) mice. The proximal half of mouse small intestine was isolated and washed with ice-cold PBS until luminal contents were cleared. The intestine were opened longitudinally, villi scraped off and cut into ~2mm pieces. These fragments were washed 5 times with PBS. Tissue pieces were incubated with 2 mM EDTA in ice-cold PBS for 30-60 minutes. Crypts were isolated from up to 10 fractions after pipetting up and down 5 times with PBS. Crypts were converted to single cells by incubating with TrypLE (ThermoFisher) at 37 °C for 20-45 minutes. Single cells were stained and DAPI-negative and Epcam-positive cells were sorted and frozen in culture media containing FBS and 10% DMSO. Cryopreserved cells were sent to Active Motif to perform the ATAC-seq assay. The cells were then thawed in a 37C water bath, pelleted, washed with cold PBS, and tagmented as previously described (Buenrostro et al. 2013), with some modifications based on (Corces et al. 2017). Briefly, cell pellets were resuspended in lysis buffer, pelleted, and tagmented using the enzyme and buffer provided in the Nextera Library Prep Kit (Illumina). Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nature Methods 10, 1213-1218 (2013). Corces, M. R., Trevino, A. E., Hamilton, E. G., Greenside, P. G., Sinnott-Armstrong, N. A., Vesuna, S., … Chang, H. Y.. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nature Methods, 14(10), 959-962 (2017). Tagmented DNA was then purified using the MinElute PCR purification kit (Qiagen), amplified with 10 cycles of PCR, and purified using Agencourt AMPure SPRI beads (Beckman Coulter). Resulting material was quantified using the KAPA Library Quantification Kit for Illumina platforms (KAPA Biosystems). Nextera Library Prep Kit (Illumina)
ENA-FIRST-PUBLIC
2021-01-15T04:09:21Z
ENA-LAST-UPDATE
2020-01-21T20:28:03Z
External Id
SAMEA6495623
INSDC center name
Dept. of Clinical and Molecular Medicine Norwegian University of Science and Technology (NTNU)
INSDC first public
2021-01-15T04:09:21Z
INSDC last update
2020-01-21T20:28:03Z
INSDC status
public
Submitter Id
E-MTAB-8718:WT_ATAC_S518
age
8
broker name
ArrayExpress
common name
house mouse
developmental stage
adult
genotype
Villin Cre - ; Lsd1 f/f
individual
518
organism part
crypt epithelium of small intestine
sample name
E-MTAB-8718:WT_ATAC_S518
scientific_name
Mus musculus
sex
male
strain
C57BL/6