organism: Danio rerio
description: Protocols: PGCs and somatic cells were isolated from Buc-GFP zebrafish strain at 3.3hpf, 4.3hpf and 24hpf via FACS. Cells were collected directly into 150ul 1.5X lysis buffer and snap frozen. Thaw cells and incubate with 20ug/ml RNase A at 37C for 1h, followed by 100ug/ml Proteinase K treatment at 55C for 3h. Phenol:choroform extraction and ethanol precipitation were performed to extract DNA and then resuspend it in water. Genomic DNA were extracted from 4500-7500 sorted somatic cells or PGCs at high, dome and 24hpf stage and digested with MspI at 37C for 3h. The fragment ends were repaired with Klenow exo at 37C for 50min. Then Methylated illumina Pair-end Adaptors were ligated to gDNA fragments using T4 DNA ligase. The bisulfite conversion were performed using Zymo Research EZ DNA Methylation Gold kit. Libraries were PCR amplified for 20 cycles using Platinum Taq DNA polymerase.
attributes.tag: ENA first public
attributes.value: 2020-12-24
attributes.tag: ENA last update
attributes.value: 2020-12-02
attributes.tag: External Id
attributes.value: SAMEA7678643
attributes.tag: INSDC center alias
attributes.value: MRC London Institute of Medical Sciences and Faculty of Medicine, Imperial College, London, UK
attributes.tag: INSDC center name
attributes.value: MRC London Institute of Medical Sciences and Faculty of Medicine, Imperial College, London, UK
attributes.tag: INSDC first public
attributes.value: 2020-12-24T04:05:28Z
attributes.tag: INSDC last update
attributes.value: 2020-12-02T17:34:07Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9858:Prim5_soma_rep1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: somatic cell
attributes.tag: common name
attributes.value: zebrafish
attributes.tag: developmental stage
attributes.value: pharyngula prim-5
attributes.tag: genotype
attributes.value: Buc-GFP
attributes.tag: sample name
attributes.value: E-MTAB-9858:Prim5_soma_rep1
attributes.tag: strain
attributes.value: AB