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DimmestP / chimera_project_manuscript

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Thoroughly check methods section #161

Closed ewallace closed 3 years ago

ewallace commented 3 years ago

We need to double-check the methods section, including disambiguating the various labware, "deepwell" or not.

@daneckaw has agreed to do this by next week (11th June). Then we should kick back over to @j-aux for another check.

ewallace commented 3 years ago

@daneckaw sent comments, @DimmestP will address Wednesday 16th afternoon.

DimmestP commented 3 years ago

@j-aux @daneckaw

Question about the Construction of chimeric reporter plasmids section of the methods.

Currently it says:

Assembled plasmids were transformed into yeast BY4741 using lithium acetate transformation (Gietz and Woods 2002), and selected in SC-URA agar plates to isolate successful transformants.

Weronika has commented:

A variant with DMSO as enhancer was used. It is described as one of options in the method paper, but it is not a part of the basic protocol.

Is this clearly explained in a protocol on protocols.io? Or does that need to be clarified here?

Also there is a reference to a supplementary table that is not there. Do we have a plan for it to be created?

DNA sequences used in this study are summarised in Supplementary table X.

j-aux commented 3 years ago

It's in the protocols.io protocol. Perhaps we change it to:

"Assembled plasmids were transformed into yeast BY4741 using lithium acetate transformation with DMSO (Gietz and Woods 2002), and selected in SC-URA agar plates to isolate successful transformants."

Though this is already an incredible amount of detail for our methods section.

DimmestP commented 3 years ago

@j-aux

Questions about the RNA measurements: Strain growth, RNA extraction, RT-qPCR, and analysis section of the methods.

Currently it says:

We summarize here a detailed protocol that is available at (CITE protocols.io).

This links with issue #96, what is the plan for citing protocols? Where are they currently? Looking at the papers that protocol.io give as examples for how to cite it's very ironic as the links have gone stale...

Also, there is another reference to the supplementary data that hasn't been added. Plan of action?

Primer sets were designed to detect the mCherry coding sequence, as well as internal reference genes RPS3, PGK1 and (plasmid marker) URA3, (see supplemental data X)

Also, are we happy to swap "24 deepwell plate" to "24-well deep well plate". And did we get them from 4titude or Starlab (as Weronika suggests)?

Finally in the second paragraph we say

The pelleted cells were thawed and individually resuspended in 400 $\mu$l of RNA binding buffer (Zymo cat #R1013-2), then transferred to 2 ml screw cap tubes containing zirconia beads, lysed using the Precellys Evolution homogeniser then pelleted by centrifugation at 12,000g for 1.5 minutes.

Is the amount of zirconia beads added and the programme used on the Precellys Evolution homogeniser available on protocol.io or do we need to add that here?

DimmestP commented 3 years ago

@j-aux

Questions about the Design of modified 3’UTRs for testing the effects of mutated motifs section of the methods.

What settings where used RNAfold to make the secondary structure figures?

Currently it says:

These [motif] positions were selected based on key design criteria (SUP FIGURE/NOTE X). Whats the plan for making the sup figure associated with this?

DimmestP commented 3 years ago

Other than the points raised above I have added all of @daneckaw comments to the materials and methods section in the branch weronika_method_comments

DimmestP commented 3 years ago

Clemence noticed that there is another supplementary table reference in the materials and methods that is actually missing. A table surmising the 3'UTRs we could have chosen as hosts for the motifs (from which we chose TSA1 and PIR1). It is referenced in Design of modified 3'UTRs for testing the effects of mutated motifs.

daneckaw commented 3 years ago

Is the amount of zirconia beads added and the programme used on the Precellys Evolution homogeniser available on protocol.io or do we need to add that here?

Amount of zirconia beads is on protocols.io, but the Precellys programme is not (at least not in the protocol I have access to) - it is only described as "EW Yeast protocol".

DimmestP commented 3 years ago

I will recreate the cumulative frequency graphs for the positions of the motifs in their native 3'UTRs to send to Jamie to create the supplementary figure explaining the motifs positions based on key design criteria.

From meeting with Jamie on 19/07/21

j-aux commented 3 years ago

Ok I'll add the following this week (from discussions with @DimmestP):

j-aux commented 3 years ago

Addressing - precellys protocol in the methods section and protocols.io protocol

I don't think its necessary to go into that much detail in the paper, and instead, we can just cite the different protocols.io protocols we use in the methods section of the paper.

ewallace commented 3 years ago

There are 4 remaining dangling references, can we fix asap?

  1. "DNA sequences used in this study are summarised in Supplementary table X."
  2. "All primer sets were thoroughly validated by serial dilution and by confirming amplicon size, sequences are available in Supplementary data X."
  3. "These positions were selected based on key design criteria (Supplementary Figure X)."
  4. "To this end, median-length 3'UTRs were extracted ... (Supplementary Table X)"