Rui:Collaborations: Difference between revisions
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>RuiLiu |
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Attached is an inventory that include what Kwonho has sent and what he has | Attached is an inventory that include what Kwonho has sent and what he has | ||
collected and has not sent. | collected and has not sent. | ||
Received on 8.10.11 | |||
#. E9.5 wildtype: 3 tubes (~250, ~250 and ~600 cells) | |||
#. E11.5 wildtype: 3 tubes (~700, ~1000, ~1000 cells) | |||
#. E13.5 wildtype-male: 2 tubes (~5000 and ~5000 cells) | |||
#. E13.5 wildtype-female: 2 tubes (~2000 and ~3000 cells) | |||
#. E13.5 mutant-male: 2 tubes (~300 and ~800 cells) | |||
#. E13.5 mutant-female: 2 tubes (~1000 and ~800 cells) | |||
===Dr. Xu Yang lab=== | ===Dr. Xu Yang lab=== |
Revision as of 17:26, 11 August 2011
Samples
Dr. Yi Zhang lab
- Sperms: ~50cell/tube; 15 tubes
- ES cells: ~50cell/tube; 15 tubes
- E9.5 bag: ~641 cells in 1 tube; ~316 cells in 1 tube.
- E11.5 bag: ~21550 cells in 1 tube.
- E13.5 bag: ~30078 cells in 1 tube (male PGCs); ~57059 cells in 1 tube (female PGCs)
1. Determine exactly what is demethylated in male pronucleus in zygotes. For this project, we need to do bisulfite sequencing before male pronucleus demethylation (demethylation start at PN3 stage, we can use sperm to mimic this) and after demethylation (PN4-5 stages, we have isolated 1000 pronuclei one by one from 1000 PN4-5 stage embryos). We can send you the samples when you are ready.
2. Understand the role of Tet1 in PGC demethylation. As you know, PGC demethylation starts at E9.5 and complete at E13.5. Therefore, we first need to find out what is demethylated in wild-type PGC by comparing the bisulfite result at E9.5 and E13.5. Then, we want to know what is not demethylated after Tet1 KO at E13.5. Therefore, meaningful sample should be wild-type PGC at E9.5 and E13.5 as well as mutant E13.5 PGC. I want to note that at E13.5, male PGC and female PGC can be distinguished. The methylation pattern of imprinted genes and sex chromosomes can be different in male and female PGC. Attached is an inventory that include what Kwonho has sent and what he has collected and has not sent.
Received on 8.10.11
- . E9.5 wildtype: 3 tubes (~250, ~250 and ~600 cells)
- . E11.5 wildtype: 3 tubes (~700, ~1000, ~1000 cells)
- . E13.5 wildtype-male: 2 tubes (~5000 and ~5000 cells)
- . E13.5 wildtype-female: 2 tubes (~2000 and ~3000 cells)
- . E13.5 mutant-male: 2 tubes (~300 and ~800 cells)
- . E13.5 mutant-female: 2 tubes (~1000 and ~800 cells)
Dr. Xu Yang lab
- GFP(+): Pancreatic lineage differentiated from hESC, NGN3+ (~50000 cells, purified with Qiagen RNeasy), 20.8ug/ml, use 1ul
- GFP(-): Pancreatic lineage differentiated from hESC, NGN3- (~50000 cells, purified with Qiagen RNeasy), 12.7ug/ml, use 1ul
- 7/7/11: Origin (?) Method (cell sorting --> spin top speed for at least 5min, done by Dae-Hwan)
Negative control (No fluorescence)--- 2 tubes (>200,000 cells each) RFP positive ------ 1 tube (~12,000 cells) GFP/RFP double positive ------ 1 tube (~55,000 cells)
Quick note
MmeI primer digestion [1]
50 cells RNAseq | procedure | samples | start | end | note |
~25 cells | RNA or lysate | Sperm vs ES cells | 5.19.11 | 5.20.11 | |
process, clone | 5.21.11 | 5.24.11 | 5.25.11 seq result | ||
Nextera library | 5.23.11 | 5.23.11 | spread out (clone?) | ||
Solexa library | 5.23.11 | 5.23.11 | amp. Not good | ||
5.26.11 | 5.26.11 | again. Not good | |||
6.1.11 | 6.2.11 | different buffs | |||
6.2.11 | 6.3.11 | another 2ug repeat | |||
6.4.11 | Kapa for NGS |
2011
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