10:00a – 12:50a

Ph Project

• Meeting with Ajaz, discuss paper revisions.
• continue preparing slides for discussion with XZ.

Meeting notes (4p – 7:30p)

To image by Confocal (top priority)

• Endogenous Ph
• Endogenous Pc
• Endogenous Psc (may not work)
• Ph-Flag
• Ph-M-Flag

figure tweaks

• increase contrast in Fig 1 F.
• Thicker scale bars on all panels in Fig 2
• Put more heavily dispersed Ph into Fig 5.
• Fig2 Panel I find example with stronger colocalize
• better Y axis label for histograms.

controls for supplement

• Supplement figure S1.
• No primary controls
• Flag in S2 cells with DAPI contrast.

Fig 5

• change figure back to Ajaz format
• Refer to supplement.
• don’t want to do review experiments to show chromatin template effects

Model for supplement

• Chromatin template important or not?
• Effect of clustering on radius gyration

Expression analysis

• Ajaz will send raw expression files.
• see if we take top 30 Pc target genes, how many increase expression vs. the average gene. Something like this.

Chromatin Project

Sequencing Prep

• Get library from Hao
• check primer concentrations
• 2x Plate PCR of library. D01 – G12
• ran 25 uL scale PCR with T7 and common primers
• pool these, add on T7-NEB adapter in 3 rounds of PCR

Lib2 probes to sequence

• F11-P3 3 replicates of probe synthesis
• F12-P3 2 replicates of probe synthesis
• G09-P1 1 probe sample
• D12-P1 1 probe sample (top left of orange box)
• all diluted 0.5 uL to 200 uL ddH2O

Poor man’s limited cycle PCR:

• Lib2X, Lib4E1, Lib4E3, F11NC, G09

Focus: birth and death of a probe in PCR

LC-PCR

1. Lib2-x1 1:32, (2 uL)
2. Lib2-x2 1:32,
3. Lib2-x1 1:32 v2. (2 uL)
4. HaoLib4 E1 (8 uL)
5. HaoLib4 E3
6. D12-P1 (2 uL)
7. G09-P1
8. F11-P3 Nc
9. Fll-P3 12-18
10. F11-P3
11. F12 P3 NC
12. F12 P3 1580
13. Hao L3-uni + L3 i1R
14. Hao L3-uni + L3 i2R
15. Hao L4 E1F + E1R
16. Hao L4 E3F + E3R

Completed limited cycle PCRs and gel extraction and cleanup.