Merge branch 'latest' of https://github.com/GallowayLabMIT/protocols …

…into latest

.github/workflows/build_docs.yaml

@@ -8,7 +8,7 @@ jobs:
     runs-on: ubuntu-latest
 
     steps:
-    - uses: actions/checkout@v2
+    - uses: actions/checkout@v4
       with:
           fetch-depth: 0
     - name: Install dependencies
@@ -18,9 +18,9 @@ jobs:
                                texlive-latex-extra latexmk tex-gyre \
                                --no-install-recommends
     - name: Set up Python
-      uses: actions/setup-python@v2
+      uses: actions/setup-python@v5
       with:
-        python-version: 3.7
+        python-version: 3.12
     - name: Install Python dependencies
       run: pip install -r requirements.txt
     - name: Build documentation and deploy

docs/bootcamp/iap/software_tips_and_tricks.rst

@@ -114,7 +114,6 @@ There are several built-in features of Zotero that make paper reading more effic
 
    .. image:: img/zotero_tablet.png
     :align: center
-|
 
 
 Better Quartzy
@@ -148,7 +147,7 @@ or in the Tampermonkey Utilities tab, you can use the **install from URL** optio
 
 .. image:: img/tampermonkey_install_from_url.png
     :align: center
-|
+
 
 Regex help
 ----------
@@ -173,7 +172,7 @@ you are using.
 
 .. image:: img/fira_code.png
     :align: center
-|
+
 
 Activate SnapGene remotely
 ----------------------------

docs/conf.py

@@ -64,7 +64,6 @@
 import rushd as rd
 import scipy
 import seaborn as sns
-from statannotations.Annotator import Annotator
 from matplotlib import ticker as mticker
 
 sns.set_theme(style="ticks")

docs/general/repairing_deli_fridge.rst

@@ -4,7 +4,7 @@ Deli fridge starter replacement
 When the deli fridge refuses to start, it is possibly due to the starter components. The starter components are responsible for getting the initial burst of energy needed to turn over the compressor motor. Luckily, both of these parts are easy to replace!
 
 Background
-==========
+----------
 The starter consists of two parts:
 
 1. A current-sensitive starter relay: `this video <https://www.youtube.com/watch?v=PRq1WPH1sRg>`__ discusses the details, but this is effectively a relay that turns on when it senses current above a certain threshold, entirely mechanically.
@@ -20,7 +20,7 @@ We can reorder the parts for relatively cheap:
 
 
 Disassembly
-===========
+-----------
 
 1. Unplug the deli fridge from the wall. Wait about 5 minutes for capacitors to discharge.
 2. Take the front grill off. It just pops off if you pull it horizontally out.
@@ -65,7 +65,7 @@ Disassembly
 
 
 Debugging
-==========
+---------
 You don't need to disconnect any wires between the capacitor and relay to test it.
 
 1. Check if the relay is bad or not. The relay switches via a metal piece that gets pulled up into the
@@ -86,7 +86,8 @@ You don't need to disconnect any wires between the capacitor and relay to test i
 If the relay resistance is fine and switches when you invert it, then try swapping the capacitor.  
 
 Assembly
-========
+--------
+
 1. After deciding which part to replace (possibly both!) connect the replacement capacitor and relay by using the existing connectors. This should not require any wire stripping; just reuse the wires with the push-on connectors.  Assemble it as in the picture. It is **important which terminals you connect the white and black wires to**, but it does not matter which orientation you attach the capacitor (it's an AC capacitor so no polarity).
 
 .. figure:: ../img/deli_starter_connections.jpg
@@ -109,6 +110,7 @@ Assembly
 
 
 Replacements:
-========
+-------------
+
 - 2023.11.10 - Capacitor
 - 2024.06.24 - Capacitor

docs/index.rst

@@ -30,6 +30,6 @@ Click `here <galloway_lab_protocols.pdf>`_ to access the PDF version.
 Contributing
 =============
 
-See the :doc:`Repository setup guide <tech/repo_setup>` if you are interested in how the continuous build system works, or you'd like to fork this repository to make it your own.
+See the :doc:`Repository setup guide <tech/ci_builder_config>` if you are interested in how the continuous build system works, or you'd like to fork this repository to make it your own.
 
 See the :doc:`Contributor guide <contributor_guide>` for a more detailed walkthrough of how to contribute to this protocols repository.

docs/plot_gallery/index.rst

@@ -21,7 +21,6 @@ and setup code that runs before any given code examples:
    import pandas as pd
    import scipy
    import seaborn as sns
-   from statannotations.Annotator import Annotator
    from matplotlib import ticker as mticker
 
    sns.set_theme(style="ticks")

docs/protocols/biochem_and_analytics/sci_ATAC_seq.rst

@@ -477,7 +477,7 @@ Transposase assembly
 
    .. note:: 
 
-      If you are doing an entire plate, then you can use :math:`Z = 3.5`μL for the sci.AD1 primers and :math:`Z = 3.0`μL for sci.AD2.
+      If you are doing an entire plate, then you can use :math:`Z = 3.5` μL for the sci.AD1 primers and :math:`Z = 3.0` μL for sci.AD2.
 
 3. Prepare the unloaded D1 dilution of Tn5 by diluting Tn5 with dilution buffer. Using the Diagenode 2 mg/mL Tn5, the dilution ratio can be at least 1:10 (e.g. 9.0 μL Tn5 diluted to 90 μL). This has been verified to work at 1:10.
 

docs/protocols/tc/cell_line_creation.rst

@@ -15,43 +15,35 @@ Cell Line Creation
 
     For CRISPR, Deon has developed a robust modRNA-based delivery method that is capable of single and multiplex HDR-based insertions.
 
-    For TALEN, (insert here Chris) 
-
-
 Each method possesses their unique cell plating, transfection, and selection timelines. After the selection period, the methods converge on universal enirichment and single-cell cloning protocols (ie., limiting dilution or cell sorting) 
 
 
-PiggyBac (Sneha)
-~~~~~~~~~~~~~~~~
-Day -1
+PiggyBac
+--------
+Day 0
 ~~~~~~
-Seed your cells such that they will be around 30-40% confluent on the next day. There is an
+Seed your cells such that they will be around 20-30% confluent on the next day. There is an
 extended selection outgrowth period required, which means we should start with lower confluence.
 Recommended rough seeding counts are:
 
 =========       ============================
-Cell type       Seeding amount (per 96 well)
+Cell type       Seeding amount (per 24 well)
 =========       ============================
-293T            12.5k cells
-U2-OS           7.5k cells
+293T            40k cells
+U2-OS           10k cells
 =========       ============================
 
-Day 0
+Day 1
 ~~~~~~
-Follow the step for the cell line type to generate
-    - PiggyBac
-        - Co-transfect the PiggyBac plasmid alongside your plasmid containing PB recognition sites at a 3:1, template:transposase mass ratio.
-    - Crispr
-        - Co-transfect the Crispr/guide plasmid alongside your plasmid containing locus-specific recognition sites at a 1:1 mass ratio.
-    - TALEN
-        - Co-transfect the TALEN-R and TALEN-L plasmids alongside your plasmid containing locus-specific recognition sites at a 1:1:1 mass ratio.
+- Co-transfect the PiggyBac plasmid alongside your plasmid containing PB recognition sites at a 3:1, template:transposase mass ratio.
+- If you are doing markerless integration, then co-transfect a plasmid containing your resistance gene and an unused fluorescent marker.
 
 .. note::
     When transfecting, leave some untransfected wells where you just add the KO-DMEM/PEI master mix, without plasmids.
     This allows you to tell if your selection is working as expected, and also gives you a proxy for how much PEI-mediated
     cell death you are seeing.
 
-Day 1
+Day 2
 ~~~~~~
 Media change into selection media. Tested concentrations for PiggyBac are:
 
@@ -62,16 +54,24 @@ Cell type   Puro
 U2-OS       0.25 μg/mL (40,000x)
 =========   ====================
 
-Day 2-4
-~~~~~~~
-Continue selecting the cells until decent cell death occurs, or the cells are too dense.
+Day 4
+~~~~~
+Replace the selection media and continue selecting until sufficient cell death occurs.
 
 .. note::
     Puromycin at the given concentration should decidedly kill your untransfected wells.
     If you do not see sufficient cell death, try using a different aliquot.
 
+Day 7
+~~~~~
+Passage the remaining cells to 6-well scale.
+
+Day 9
+~~~~~
+Sort!
+
 CRISPR (Deon)
-~~~~~~~~~~~~~
+-------------
 Day -1
 ~~~~~~
 Deon recommends CRISPR at 12-well scale, as this is the smallest scale that yielded a workable number of surviving cells after multiple transfections, CRISPR genotoxicity, and drug selection.  
@@ -129,7 +129,7 @@ Remove selection and let cells recover/expand. During your next passage, take a
 
 
 TALENS (Chris)
-~~~~~~~~~~~~~~
+--------------
 Day -1
 ~~~~~~
 Seed your cells such that they will be around 30-40% confluent on the next day. There is an
@@ -351,4 +351,4 @@ RMCE integration of payloads into V2 line
 
 
 Day -1
-~~~~~~
+-------

docs/protocols/tc/ipsc_transfection.rst

@@ -29,6 +29,8 @@ Empirically determined reagent amounts to use:
 =============================================================================================================================================== ==========================================
 
 .. note:: Not all reagent amounts have been rigorously optimized. 
+.. note:: These amounts can be scaled  to various plate sizes and DNA amounts. 
+.. note:: The protocol for Lipofectamine 3000 can be applied to transfection of Jurkat cells in suspension at low efficiency (1-3%).
 
 
 Protocol

docs/protocols/tc/tc-basics/index.rst

@@ -14,4 +14,6 @@ TC Basics
    platE_MEF_reprogramming
    human_reprogramming
    glass_slide
+   murine_bone_marrow
+   splenocyte
    *