Rui:Collaborations

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Revision as of 18:49, 3 November 2011 by >Sam Chiang (→‎Dr. Xu Yang lab)
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Samples

Dr. Yi Zhang lab

Wt mouse samples

  1. Sperms: ~50cell/tube; 15 tubes
  2. ES cells: ~50cell/tube; 15 tubes
  3. E9.5 bag: ~641 cells in 1 tube; ~316 cells in 1 tube.
  4. E11.5 bag: ~21550 cells in 1 tube.
  5. E13.5 bag: ~30078 cells in 1 tube (male PGCs); ~57059 cells in 1 tube (female PGCs)

PGC samples

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

  1. . E9.5 wildtype: 3 tubes (~250, ~250 and ~600 cells)
  2. . E11.5 wildtype: 3 tubes (~700, ~1000, ~1000 cells)
  3. . E13.5 wildtype-male: 2 tubes (~5000 and ~5000 cells)
  4. . E13.5 wildtype-female: 2 tubes (~2000 and ~3000 cells)
  5. . E13.5 mutant-male: 2 tubes (~300 and ~800 cells)
  6. . E13.5 mutant-female: 2 tubes (~1000 and ~800 cells)

Human ES differentiation samples

Project: perform RNA-seq to compare the gene expression during endoderm differentiation of human ES cells. 
Analyze ES, definitive endoderm, and pancreatic progenitor cells by RNA-seq. 
ctr/0d, B1/0d, B3/0d, ctr/2d, B1/2d, B3/2d. 
one gene plays an important role for definitive endoderm differentiation. 
compare the gene/non-coding RNA expression during the first stage differentiation, in control and knock-down cells.

Received on 9.15.11, 1000c/tube

  1. . ctr/0d
  2. . B1/0d
  3. . B3/0d
  4. . ctr/2d
  5. . B1/2d
  6. . B3/2d

Dr. Xu Yang lab

  • 6/27/11:
  1. GFP(+), Pancreatic lineage differentiated from hESC, NGN3+ (~50000 cells, purified with Qiagen RNeasy), 20.8ug/ml, use 1ul
  2. GFP(-), Pancreatic lineage differentiated from hESC, NGN3- (~50000 cells, purified with Qiagen RNeasy), 12.7ug/ml, use 1ul
cDNA library: [1]
RNAseq library: [2]
RNAseq run: [3]
  • 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)
  1. RFP positive ------ 1 tube (~12,000 cells)
  2. GFP/RFP double positive ------ 1 tube (~55,000 cells)
cDNA library:[4] 
RNAseq library: [5]
RNAseq run: [6]
  • 9/28/11: 3 samples from Dr. Xu Yang lab by Alice, RNA samples from sorted neural stem cells from nestin-rfp knockin hESCs.
  1. Low is rfp low
  2. high rfp high
  3. fetal NSCs are the human fetal cells.
cDNA library:[7]
RNAseq library: 
RNAseq run:

Quick note

MmeI primer digestion [8]

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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