1. By Keisha Persaud

2. About Gametes are totipotent Epigenetic: gene switches off and on
Goal: identify the what genes can transform PGC to gonocyte
Extensive epigenetic reprogramming around E10.5-E11.5
Include: genome-wide loss of 5-methycytosine (5mC)
Investigate the relationship between 5-methylcytosine (5mC) and 5- hydroxymethylcytosine (5hmC)
5hmC involved in DNA demethylation in PGC ]

3. Methods
Liquid Chromatography coupled with mass spectrometry (LC-MS/MS)
Whole-genome bisulfite sequencing (WGBS)
AbaSI endonuclease digestion coupled with sequencing (Aba-seq)

4. whole-GENOME BISULFITE SEQUENCING
Provides information about the combined level of 5mC and 5hmC
QIAamp DNA Micro Kit (Qiagen)
Total DNA isolated from 10,000 sorted PGC
Sonicator (Covaris)
DNA Fragmentation
Libraries prepared using NEBNext Library Prep protocol with methylated adaptor

5. whole-GENOME BISULFITE SEQUENCING
Bisulfite conversion done using Imprint Modification Kit
PCR done for 16 cycle using NEXTflex Bisulfite-Seq Kit
Illumina sequencing master mix
NEBNext Library Prep Universal and index primers
Library purified using AMPure XP beads.
Library sequenced on Illumina HiSeq2000

6. Aba-ENDONUCLEASE DIGESTION AND SEQUENCING
Robust site specific quantification and accurate genome-wide comparison of 5hmC level
within sample
Between sample combined with LC-MS/MS
Total DNA was isolated from 10,000 sorted PGCs using QIAamp DNA Micro Kit
Genomic DNA was glycosylated and then digested using AbaSi enzyme
Biotinylated P1 adapters were ligated onto AbaS1 digested DNA
Fragmented using sonicator (covaris)

7. Aba-ENDONUCLEASE DIGESTION AND SEQUENCING
Sheared P1-ligated DNA was then captured by mixing with Dynabeads
NEBnext END Repair Module and NEBnext dA-tailing Module repair and dA-tailing
20 º C and 37 º C respectively for 30 min
DNA amplified using Phudion DNA polymerase, forward and reverse primer for 16 cycles
Libraries were puried using AMPure XP beads
Sequence on Illumina HiSeq 2000 instrument

8. results
LC-MS/MS
5mC
Stable between migratory (E9.5) and early gonadal PGC (E10.5)
Reduction of 5mC between E10.5 and E11.5
Limited DNA demethylation between E11.5 and E13.5
Figure 1 | 5mC and 5hmC dynamics during epigenetic reprogramming.
Individual 5mC

9. results LC-MS/MS 5hmC Constant between E9.5 and E13.5
One magnitude lower than 5mC at E 10.5
PGC lower than in embryonic stem
Figure 1 | 5mC and 5hmC dynamics during epigenetic reprogramming.
Individual 5mC 5hmC (b, right)

10. results WGBS loss of combined 5mC and 5hMC between E10.5 and E11.5
DNA demethylation between E11.5 AND E12.5
Consistent with LC-MS which is seprete
This is combined
xtended Data Figure 3 | Further analysis of 5mC and 5hmC dynamics in PGCs. a, Combined levels of 5mC and 5hmC as determined by WGBS (left) or levels of 5hmC as determined by Aba–seq (right) at various features within the uniquely mapped part of the genome in PGCs between E10.5 and E12.5. For box plots, the upper and lower hinges correspond to the first and third quartiles, the centre line corresponds to the median, and
the maxima and minima correspond to the highest or lowest value within 1.5 × the inter-quartile range, respectively.

11. results Aba-seq 5hmC global levels are lower at E10.5 PGC than mESCs
5hmC localization in PGC is similar to ES cells
b, Levels of 5hmC (ascertained using Aba–seq) at various regulatory elements
in E10.5 PGCs (left) or E14 mouse ES cells15 (right). P values are based on ANOVA and Dunnett’s post hoc test. For box plots, the upper and lower hinges correspond to the first and third quartiles, the centre line corresponds to the median, and the maxima and minima correspond to the highest and lowest value within 1.5 × the inter-quartile range respectively

12. CONCLUSION During reprogramming both 5mC and 5hmC are lost in PGCs
5hmC has a gradual decrease
5hmC is a dynamic mark in PGCs

13. PROS AND CONS PROS CONS Multiple approaches Unorganized
Comparison between male and female PGC
Method was hard to follow – in terms of cell culture

14. Reference
Hill, Peter W. S. et al. “Epigenetic reprogramming enables the primordial germ cell-to-gonocyte transition.” Nature (2018).