1. Reduced Representation Bisulfite Sequencing to identify targets of Dnmt4 during zebrafish hematopoietic stem cell development Aniket V Gore: Isolation of DNA and RNA from FACS sorted HSCs Valya Russanova: Bisulfite library preparation MGL team : Bisulfite sequencing and analysis

2. Definitive Hematopoiesis in Zebrafish 32-36 hours post fertilization HSCs develop from the dorsal aorta True HSCs capable of generating all blood lineages The original pool of HSCs Conserved across all the vertebrates AGM-Aorta, Gonad, Mesonephros region

3. Schematic of HSC Egress From the Dorsal Aorta Notochord 32-34hpf Dorsal Aorta Cardinal Vein Boisset J et al., Bertrand J et al and Kissa and Herbomel, Nature 464, 2010

4. Notochord Dorsal Aorta Cardinal Vein Dnmt4 is Expressed by Hemogenic Endothelium

5. NH 2 N N O N N O DNA methyltransferase Addition of -CH 3 at position 5 of cytosine cytosine 5-methylcytosine DNA methyltransferase Dnmt CH 3

6. C m G C m G C C A A G G C m G C m G T G C m GC G C G C C A A G G C G C G T G C G Methylated DNAUnmethylated DNA Bisulfite Treatment and Sequencing C G C G C C A A G G C G C G T G C GT G T G T T A A G G T G T G T G T G Bisulfite sequencing: One of the ways to check DNA methylation

7. Exon1 CGI 62% 8% 277bp, 23 CpG Con MO Dnmt4 MO Cmyb gene body methylation and expression is regulated by Dnmt4 qRT-PCR Bisulfite Seq

8. 1: gene expression changes, RNA seq from total RNA isolated from control and dnmt4 deficient HSCs To study genome wide changes in DNA methylation and gene expression 2:DNA methylation changes, Reduced representation by bisulfite sequencing from DNA isolated from control and dnmt4 deficient HSCs

9. Isolation of GFP/RFP double positive HSC population From the transgenic line

10. RFP ONLY Endothelial cells RFP/GFP Double positive HSCs Marker 1 Marker 2 Marker 3 Con 1 Con 2 RT-PCR on FACS sorted cells from WT flk>mApple; cmyb>GFP 48 hpf embryos Marker gene expression showed enrichment of HSCs in double positive cell population Two sets of DNA and RNA for GFP, RFP, dark and Double positive cells HSC markers

11. 1: Cas9 in vitro synthesized RNA (Control) 2: Frozen cells, Trizol, phenol chloroform 3: Frozen cells, Trizol, phenol chloroform, DNAseI, Phenol chloroform, Phase lock 4: Frozen cells, Qiagen microRNA isolation kit 5: Live cells, Zymo DNA/RNA kit 6: Live cell, Zymo DNA/RNA kit, DNAseI treatment Different techniques to isolate good quality total RNA

12. 1: Con MO Red endothelial cells 2: Con MO double positive HSCs (195ng) (86,383 cells) 3: dnmt4 MO Red endothelial cells 4: dnmt4 MO double positive HSCs (150ng) (42,712 cells) Cells isolated from flk>mApple; cmyb>GFP embryos @48 hpf and DNA and RNA isolated from The same cells using Zymo Duet Kit

13. Sample 3: Con MO double positive HSCs (Total 62.5ng) Sample 4: dnmt4 MO double positive HSCs (Total 50ng) From Valya Total genomic DNA isolated from the same sorted cells Analyzed by picogreen for concentration And by gel for quality Con MO RFP Dnmt4 MO RFP Con MO double Dnmt4 MO double