title: Tex15_KO_2
organism: Mus musculus
description: Protocols: CD9+ spermatogonia were sorted from P14 testes as previously described14 with minor alterations: 100 μg/ml DNase was added together with foetal calf serum to stop the trypsin digest and the cell suspension was further incubated for 3 min at 32 ˚C to facilitate complete degradation of released DNA. Cells were blocked with Fc block (anti-CD16/32, clone 93, eBioscience, 1:50), followed by labelling with anti-CD45 (clone 30-F11, eBioscience, 1:200) and anti-CD51 (clone RMV-7, Biolegend, 1:50) biotin conjugated antibodies. Cells were then stained with anti-CD9APC (clone eBioKMC8, eBioscience, 1:200), anti-cKitPE-Cy7 (clone 2B8, eBioscience, 1:1600), streptavidinV450 (BD bioscience, 1:250) and 1μg/ml DAPI and sorted into medium on a BD Aria II sorter. Sorted cells were pelleted for 5 min at 500 g and snap frozen in liquid nitrogen. E16.5 gonocytes were FACS purified from Miwi2tdTom/+ foetal testes by dissecting the testes in a drop of goni-mem (DMEM (Life Technologies) supplemented with penicillin-streptomycin (Life Technologies), NEAA (Life Technologies), sodium pyruvate (Life Technologies) and sodium lactate (Sigma)) and digestion in 0.25 % Trypsin-EDTA (Gibco) at 37 ˚C for 10 min Digestion was stopped by addition of 20 % foetal calf serum (FCS) and cells pelleted for 5 min at 100 g. The pellet was treated with 10 μl 5 mg/ml DNase (Sigma) for 2 min and cells rigorously resuspended in PBS containing 2 % FCS by pipetting 50 times. Cells were sorted on a BD Aria Fusion sorter into PBS, lysed in Qiazol (Qiagen) and snap frozen in liquid nitrogen. DNA from FACS purified P14 spermatogonial stem cells was isolated by proteinase K digest (10 mM Tris-HCl pH8 , 5 mM EDTA , 1 % SDS, 0.3 M Na-acetate, 0.2 mg/ml proteinase K) overnight, followed by two rounds of phenol/chloroform/isoamylalcohol (25:24:1, Sigma- Aldrich) extraction and one round of chloroform extraction. The DNA was precipitated at -20˚C after addition of 1/10 volume 3 M Na-acetate, 10 μg linear acrylamide (Invitrogen) and 1 volume of isopropanol, washed two times and solubilized in 5 mM Tris pH 8. Methyl-seq libraries were prepared using the NEBnext Enzymatic Methyl-seq kit (NEB) according to the manufacturer's instructions and sequenced by Illumina sequencing.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-06-17T17:05:21Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-05-19T16:37:50Z
attributes.tag: External Id
attributes.value: SAMEA6848077
attributes.tag: INSDC center name
attributes.value: University of Cambridge, Cancer Reserach UK - Cambridge Institute
attributes.tag: INSDC first public
attributes.value: 2020-06-17T17:05:21Z
attributes.tag: INSDC last update
attributes.value: 2020-05-19T16:37:50Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9090:Tex15_KO_2
attributes.tag: age
attributes.value: 14
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: juvenile stage
attributes.tag: genotype
attributes.value: Tex15 -/-
attributes.tag: individual
attributes.value: 2
attributes.tag: organism part
attributes.value: testis
attributes.tag: sample name
attributes.value: E-MTAB-9090:Tex15_KO_2
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: male
attributes.tag: strain
attributes.value: C57BL/6N