Alistair's Lab Notebook

9:30 am – 9:15 pm

MERFISH

• working on aligning data
• current pentanotch no quadview approach requires different approach to beads
• try multilabeled beads (homemade tetraspec with 750 dyes)
• try using labeled cells

Chromatin meeting with XZ

• priority on PcG knockdown
• use immunostain of protein to quantify protein efficiency KD
• try to increase KD efficiency of individual KDs.
• keep focus on multi component KD for imaging work

Apps and Conferences

• finalize DF files, submit to Matt
• send signature form for internal signatures
• working on slides for SDB meeting
• getting a few things for ML party at SDB

To order

• secondary sequences fused to common sequence
• more RNAi primers
• more PCR primers

9:00 am – 12:00 am

Goals

• MERFISH report requested by XZ. try to get L15 data analyzed for Wed
• Chromatin report requested by XZ. put together slides for this meeting
• finish slides for SDB meeting
• proof-read DF application and prepare to mail.
• Plate and fix RNAi knockdown cells (5 pm).

Cell culture

• BG3 cells arrived
  • lots of cells: moved to 75cm2 flask + 2 small 25cm2 flask after the settled
  • need insulin for growth media. Ordered this today, hopefully it arrives soon.
• old Hs Kc cells too dense.
• Plated new Kc cells. allowed 1 hr to attach and settle and then Heat Shocked for 2 hours at ~37C.

Knockdown experiments

prepped RNA

• sample order
  1. mock
  2. PPES
  3. Psc
  4. Ph-p, Ph-d
  5. Pho
  6. Su(z)2
• extracted RNA, using just 1.5 mL of cells and Qiagen Midi kit (350 uL lysis buffer)
• concentrations in the 100-300 ng/uL range

plated cells

• 500 uL per coverglass of cells. Reasonable confluence, get 6 coverslips + 1 mL for RNA extraction

HS experiments

• 2 hour heat shock treatment (see above)
• isolated / prepped RNA
• concentration in the 500 – 700 ng/uL range

MERFISH

• found bug in previous normalization (was ID’ing TCEP movies wrong)
• fixed normalization
• default decoding still doesn’t work.

Presentation

• proof-reading and editing DF app
• working on slides for SDB
• contacting Levine lab members for get together

9:00 am – 5:15 pm,

Looking at HS data

HS transcription

Data from Gonsalves et al 2011 PLoS ONE paper.

20 min probably not enough time for a lot of degradation — don’t see much down regulation
See the few HS genes (like hsp70) which are supposed to be rapidly up-regulated come on.

Let’s look at transcription factors and PolII binding instead.

ChIP data (from Li et al 2015 Mol Cell paper from Corces lab)

Large active locus:

so far looking good — active marks are being depleted, PolII drops.

What about at some heat shock loci?

Summary from Li et al 2015 paper:

A closer look at the data

• Top panel
  • red HS-PolII
  • magenta HS-H3K4me3
  • magenta dash HS-H3K4me1
  • blue NT-PolII
  • cyan NT-H3K4me3
  • cyan dash NT-H3K4me1
• middle panel
  • red: HS-Pc
  • blue: NT-Pc
• lower panel
  • genes, color coded by expression.

This is more troubling, PolII largely goes down, even though I validated that expression of the genes increases according to the Gonsalves et al 2011 PLoS ONE paper

what about a silent locus?

• here’s ANT-C. This is also confusing. expression clearly increases, but so does Polycomb

MERFISH

 <ul>
 <li>discussion with Jeff</li>
<li>should start the L15 data processing (first full dataset in a while acquired over the weekend).</li>
 </ul>

Conference stuff

booking transport for SDB meeting

• snowbird registration number 2C019O
• arrive on Thursday, July 9th, 2:38 pm
• pickup at 9am on Monday.
• 1-800-232-9542
  • Select option 3- Accounting assistance on an existing reservation from the menu. Give agent your Snowbird reservation
    number to order shuttle at reduced rate.
• canyon transportation van, outside when you arrive
• van phone: 1-800 255 1841

Writing

• working on DF application

Chromatin project

• aliquoted probes for all the blue internal regions.

4:00 pm – 6:15 pm

Goals

• compare wt vs HS size stat data
• get probes for staining RNAi samples.

Chromatin data analysis

• issue with code and mismatch in drift data BB reports now corrected (should check visually to confirm).
• compare BB new heat-shock image data with wt
• looks promising, 1 locus is behaving funny – should check which genes are in which locus and how they respond.

Chromatin Probes for looking at RNAi results

• L4E17 and L4E18 were made with P2 back in august
• 12-18-13 also has what I think are good probe sets for all the pieces of BX-C and ANT-C in multiple colors. Should check images.

10:05 am – 8:00 pm

RNAi

Re-analyzed data

• issues with row/column labels confused analysis
• round 1 results:
  
• repeated qPCR experiments with higher concentration cDNA
• round 2 results:
  
• looks promising!
• to do next
  • proceed with staining some cells for Pc to validate at single cell level
  • make more cDNA from isolated total RNA for deep sequencing.

New RNAi setup

• Plate 1
  • row 1: Th 30 ug, Mock, Psc 60 uG
  • row 2: Pc, Psc, Esc, Scm 30ug each, Ph-p + Ph-d, 40 ug each, Su(Z)2 30 ug
• Plate 2
  • row 1: Psc 60 ug, Psc 60 ug, Psc 60 ug
  • row 2: Ph 30 ug, mock, mock

Applications

• rewriting text of DF application
• working on figures for DF application

Conferences

• finished registration and payment for EMBO Nuclear structure meeting

MERFISH

• analyzing Guiping’s new data.
• 512×512 36 bits is running very slowly.
• its the upsampled CorrAlign command that kills the run speed.
• sped up CorrAlign
  • doesn’t need to align every bit, just every hybe
  • doesn’t need whole image, should work with just the center field of view (may have trouble, we’ll see).
• is this really supposed to be 36 bit data?
• where’s the codebook?

8:45 am – 5:50 pm, 8:45 pm – 10:50 pm

Goals

• finish letter for DF application

Coding

• small fix to accelerate FiducialDriftCorrection function

Dropbox failure

Yesterday I selectively sync’d my dropbox account on Tuck to only update the public folder and nothing else. The goal was to save space on Tuck’s harddrive. This didn’t save any space yet, it just stopped sync’ing these folders. So next I deleted the un-sync’d folders. I did this through remote-desktop connection to Tuck mediated by my primary desktop (Monet).
Today I noticed that all my files on Monet were deleted (Monet actually had dropbox uninstalled). All the files on Morgan except public were also deleted, and logging into dropbox online all the files were moved to trash despite the selective sync.
On further research, the original symptom isn’t supposed to happen: when I chose selective sync, dropbox is supposed to remove the folder from that computer immediately (not just stop synching). See this note from the dropbox help.

RNAi experiments: QPCR quantification

Well assignments Top row ‘A1-> H1’ (actually the way the label it on the plate its H1-A1, but I’ve renamed it so the numbers and letters match the spreadsheet number and letter column/row labels).

Setup

• make primer dilution and mix forward and reverse primers at a final concentration of 50 uM
• To each of 4 top left wells, add 8.4 uL corresponding cDNA and 75.6 uL of water
• move 21 uL of this mix to each of the top 4 rows.
• move 11 uL from each of the top 4 rows to the second 4 rows
• remove 9 uL from each of the second 4 rows and set aside. Add 9 uL ddH2O
• move 1 uL from each of the second 4 rows after dilution to the bottom 4 rows. Add 9 uL ddH2O
• The cDNA dilutions are now complete.

Master mix

• per well: 25 uL Phusion Master, 2.5 uL SyberGreen, 6.5 ddH2O
• 4*(12+1) wells multiplier is 52.
• 1300 uL Phusion
• 130 uL 20x SyberGreen
• 338 uL ddH2O

Final setup

• Duplicate all wells except the first row in the neighboring 4 columns.

Results:

Repeat at higher concentration

• per well:
  • 12.5 uL Phusion hot start master mix
  • 1.25 uL 20x EvaGreen
  • 5.75 uL ddH20
• 20x master
  • 250 uL Phusion hot start master mix
  • 25 uL 20x EvaGreen
  • 115 uL ddH20
  • divide into 19.5 uL per well
• to each add well, add:
  • 2.5 uL primer mix
  • 3 uL cDNA
• 16 total tubes, 4 RNAi conditions and 4 gene-PCR primers
• running overnight

9:15 am – 12:15 am

Writing & grant stuff

• working on DF applications
• sorting out agreement logistics with DR and BWF

Coding

• rewrote FiducialDriftCorrection function to allow for more drift even when beads are dense

STORM2

• issues: with 560 shutters file (see email)
• no cylindrical lens for 3D yet. Ye has parts.

9:45 am – 7:30 pm, 8:30 pm – 10:00 pm

RNAi

• setup RNase digestions
• fixed cells (probably too late for these guys, actually started Tuesday night).

Primer design

• would be useful to do by batch in matlab rather than manually
• looking up existing matlab functions for getting gene sequences etc.
• troubleshooting
  • getgenbank returns over a quarter million features when called on Drosophila abd-B. (32 Mb of sequences mapping to unassembled parts of the chromosome). actually it’s all of Chr3R, starting from heterochromatic bases
  • need to use the nucleotide search option in genbank, search for gene name and melanogaster, select transcript, get accession number of that.
• exploring Jeff’s DesignPrimers wrapper for primer3.
• installed primer3 (best to get the windows binary version)
• Completely awesome (as expected). Could use a Tm choice feature?
• primers designed for
  'Abd-B','NM_001275719';
'abd-A','NM_169733';
'Ubx','NM_169728';
'en','NM_078976';
'Antp','NM_206445';
'Dfd','NM_057853';
'lab','NM_001260024';
'Pc','NM_079475';
'Psc','NM_001299439';
'Esc','NM_058083';
'ph-p','NM_001297873';
'ph-d','AM943679';
'pho','NM_166827';
'Sce','NM_058161';
'Su(z)2','NM_079002';
'Scm','NM_001260077';

Orders

• ordered BG3-cl2 cells
• ordered NEB sequencing kit (to replace elements I’ll use from JM/SW stocks)
• ordered primer plate (all Fwd primers, then all rev primers)

To do

• set up new RNAi experiments
• design primers