alias: E-MTAB-6785:Tet2Tet2CM_EpiLC_RRBS_rep1_s
title: Illumina HiSeq 1500 sequencing; Tet1 controls maintenance DNA methylation via the regulation of Dppa3
design: Tet1 controls maintenance DNA methylation via the regulation of Dppa3
library_construction_protocol: Cells were collected by trypsinization To differentiate naïve ESCs into Epiblast-like cells (EpiLCs), flasks were first pre-treated with Geltrex (Life Technologies) diluted 1:100 in DMEM/F12 (Life Technologies) and incubated at 37 °C overnight. Naïve ESCs were plated on Geltrex-treated flasks in defined medium containing 10 ng/mL Fgf2 (R&D Systems), 20 ng/mL Activin A (R&D Systems) and 0.1× Knockout Serum Replacement (KSR) (Life Technologies). Media was changed after 24 h and EpiLCs were harvested for RRBS and RNA-seq experiments after 48 h. For the generation of Tet1, Tet2, and Tet1/Tet2 catalytic mutants, specific gRNAs targeting the catalytic center of Tet1 and Tet2 were cloned into a modified version of the SpCas9-T2A-GFP/gRNA (px458;(Ran et al., 2013), Addgene plasmid #48138), to which we fused a truncated form of human Geminin (hGem) to SpCas9 in order to increase homology-directed repair efficiency (Gutschner et al., 2016).  A 200 bp ssDNA oligonucleotide harboring the H1652Y and D1654A mutations and ~100 bp of homology to the genomic locus was synthesized (IDT, Coralville, IA, USA). For targetings in wild-type J1 ESCs, cells were transfected with a 4:1 ratio of donor oligo and Cas9/gRNA construct. Positively transfected cells were isolated based on GFP expression using fluorescence-activated cell sorting (FACS) and plated at clonal density in ESC media 2 days after transfection. Cell lysis in 96-well plates, PCR on lysates, and restriction digests were performed as previously described (Mulholland et al., 2015). Tet1 catalytic mutation was confirmed by Sanger sequencing. For insertion of the HALO or eGFP coding sequence into the endogenous Dppa3 and Uhrf1 loci, respectively, Dppa3 and Uhrf1 specific gRNAs were cloned into SpCas9-hGem-T2A-Puromycin/gRNA vector, which is a modified version of SpCas9-T2A-Puromycin/gRNA vector (px459;(Ran et al., 2013), Addgene plasmid #62988) similar to that described above. To construct the homology donors plasmids, gBlocks (IDT, Coralville, IA, USA) were synthesized containing either the HALO or eGFP coding sequence flanked by homology arms with ~200-400 bp homology upstream and downstream of the gRNA target sequence at the Dppa3 or Uhrf1 locus, respectively, and then cloned into the NruI site of pUC57-GentR via cut-ligation. ESCs were transfected with equimolar amounts of gRNA and homology donor vectors. Two days after transfection, cells were plated at clonal density and subjected to a transient puromycin selection (1 ug/mL) for 40 h. After 5-6 days, ESCs positive for HALO or eGFP integration were isolated via fluorescence-activated cell sorting (FACS) and plated again at clonal density in ESC media. After 4-5 days, colonies were picked and plated on Optical bottom µClear 96-well plates and re-screened for the correct expression and localization of eGFP or HALO using live-cell spinning-disk confocal imaging. Cells were subsequently genotyped using the aforementioned cell lysis strategy and further validated by Sanger sequencing(Mulholland et al., 2015).  Column purification For RRBS, genomic DNA was isolated using the QIAamp DNA Mini Kit (QIAGEN), after an overnight lysis and proteinase K treatment.  RRBS library preparation was performed as described previously (Boyle et al. 2012), with the following differences: bisulfite treatment was performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research Corporation) according to the manufacturer's protocol.
identifiers.namespace: Faculty of Biology Department II Ludwig Maximilians University Munich
identifiers.id: E-MTAB-6785:Tet2Tet2CM_EpiLC_RRBS_rep1_s
attributes.tag: Experimental Factor: cell type
attributes.value: epiblast-like cell
attributes.tag: Experimental Factor: genotype
attributes.value: Tet1Tet2CM