organism: Mus musculus
description: Protocols: Cells were extracted from lungs and subsequently sample was enriched for immune cells using CD45 microbeads from Miltenyi, and with flow cytometry. H3K27me3 and H3K27ac were profiled from sorted DC using modified CUT&RUN protocol.(Skene and Henikoff 2017) Nuclei were extracted from 200.000 sorted cells. Cells were collected by centrifugation (500g, 5min 4ºC) and gently resuspended in 50ul lysis buffer containing 10mM Tris pH 7.4, 10mM NaCl, 3mM MgCl2, 1% BSA, 0.1% Tween-20, 0.1% IGEPAL, 0.01% Digitonin (Promega), 1mM DTT, 1x cOmplete protease inhibitor and incubated on ice for 3min. Then we added 50 ul of wash buffer containing 10mM Tris pH 7.4, 10mM NaCl, 3mM MgCl2, 1% BSA, 0.1% Tween-20, 0.1%, 1mM DTT, 1x cOmplete. Nuclei were collected by centrifugation (500g, 5min 4ºC) and resuspended in 100 µl room temperature NE Buffer. 5µl/sample Concanavalin A (ConA) beads (Epicypher) were activated by washing twice with 100ul/10ul of beads of cold Bead Activation Buffer (20mM HEPES pH 7.9, 10mM KCl, 1mM CaCl2, 1mM MnCl2). Beads were resuspended in 10µl/sample cold Bead Activation Buffer. Nuclei were incubated with concanavalin beads at room temperature for 10 min. Beads were placed on the magnet, supernatant was removed and nuclei were resuspended in 50 µl of cold wash buffer (20mM HEPES pH 7.5, 150mM NaCl, 0.5mM spermidine, 1x cOmplete, 2mM EDTA). 0.5ul of primary antibody (H3K27me3 antibody, C36B11, Cell Signalling; H3K27ac antibody, D5E4, Cell Signalling; Isotype control antibody, DA1E, Cell Signalling) was added to each sample and incubated at the nutator at 4C overnight. Beads were placed on the magnet, supernatant was removed and nuclei were resuspended in 50ul of digitonin buffer (20mM HEPES pH 7.5, 150mM NaCl, 0.5mM spermidine, 1x cOmplete, 0.01% Digitonin, 2mM EDTA) and 0.5µg of anti-rabbit secondary antibody, epicypher was added, mixed and incubated for 30min at room temperature. While beads were on magnet they were washed with 200 µl of cold digitonin buffer twice and then resuspended in 50ul of high salt digitonin buffer (20mM HEPES pH 7.5, 300mM NaCl, 0.5mM spermidine, 1x cOmplete, 0.01% Digitonin, 2mM EDTA), 2.5 µl of pAG-Tn5 (Epicypher) to each sample, vortexed and incubated for 1h at room temperature. Nuclei were then washed twice with high salt digitonin buffer and 50µl of cold tagmentation buffer was added (20mM HEPES pH 7.5, 300mM NaCl, 10mM MgCl2, 0.5mM spermidine, 1x cOmplete) and incubated for 1h at 37ºC. After tagmentation, nuclei were washed with 50µl of 10mM TAPS pH 8.5 with 0.2mM EDTA. To release DNA fragments, 5ul of 10mM TAPS pH 8.5 with 0.1% SDS was added to nuclei and incubated for 1h at 58C. Later, 15ul of 0.67% Triton-X were added to neutralise SDS. I nto 20ul of sample 25ul of non-hot start CUTANA High Fidelity 2x PCR Master Mix and 2ul each of i5 and i7 primers (Illumina) were added and incubated at 58ºC for 5 min, at 72ºC for 5 minutes, at 98ºC for 45 minutes and then 18 cycles of 15s 98ºC and 10s 60ºC, followed by final extension at 72ºC for 1 min. Libraries were cleaned up using 1.3x AMPure beads as per manufacturer's recommendations and eluted in 15ul of TE buffer.
identifiers.namespace: BioSample
identifiers.id: SAMEA12789777
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-01-01
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-01-01
attributes.tag: External Id
attributes.value: SAMEA12789777
attributes.tag: INSDC center alias
attributes.value: WIS
attributes.tag: INSDC center name
attributes.value: Weizmann Institute of Science
attributes.tag: INSDC first public
attributes.value: 2023-01-01T00:27:28Z
attributes.tag: INSDC last update
attributes.value: 2023-01-01T00:27:28Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11390:A_ic_1
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: Akita
attributes.tag: individual
attributes.value: pool of mice
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: lung
attributes.tag: sample name
attributes.value: E-MTAB-11390:A_ic_1
attributes.tag: sex
attributes.value: male
attributes.tag: strain
attributes.value: C57BL/6