organism: Mus musculus
description: 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)
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2021-01-15T04:09:21Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-01-21T20:28:03Z
attributes.tag: External Id
attributes.value: SAMEA6495621
attributes.tag: INSDC center name
attributes.value: Dept. of Clinical and Molecular Medicine Norwegian University of Science and Technology (NTNU)
attributes.tag: INSDC first public
attributes.value: 2021-01-15T04:09:21Z
attributes.tag: INSDC last update
attributes.value: 2020-01-21T20:28:03Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8718:LSD1KO_ATAC_S519
attributes.tag: age
attributes.value: 8
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: Villin Cre + ; Lsd1 f/f
attributes.tag: individual
attributes.value: 519
attributes.tag: organism part
attributes.value: crypt epithelium of small intestine
attributes.tag: sample name
attributes.value: E-MTAB-8718:LSD1KO_ATAC_S519
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: male
attributes.tag: strain
attributes.value: C57BL/6