update library prep posts

Author: zdellaert

_posts/2024-12-06-LCM-Zymo-Trio-WGBS-Library-Prep-Test.md

@@ -134,3 +134,5 @@ THIS WORKED!!! WOAH! Almost too good!!! Will maybe do another cleanup to remove
 <img src="https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/LCM_Pilot_WGBS/2024-12-15-LCM-WGBS-Trio-library-18-ReAmp.png?raw=true" height="400">
 
 Full results can be found [here](https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/2024-12-15-WGBS-Trio-Round3.pdf)
+
+These libraries were cleaned and processed with the ones from [12/12/24](https://zdellaert.github.io/ZD_Putnam_Lab_Notebook/LCM-Zymo-Trio-WGBS-Library-Prep-Test-2/). See that post for details. 

_posts/2024-12-12-LCM-Zymo-Trio-WGBS-Library-Prep-Test-2.md

@@ -162,4 +162,4 @@ For the re-amped libraries (17, 18, 32, 33), there are not big dimers, but there
 
 Full results can be found [here](https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/2024-12-16-WGBS-Cleaned-Libraries.pdf)
 
-These are done.
+These are done. Shipped to sequencing facility along with the 12/15/24 libraries on 12/17/24.

_posts/2024-12-15-LCM-Zymo-Trio-WGBS-Library-Prep-Test-3.md

@@ -94,15 +94,15 @@ Full results can be found [here](https://github.com/zdellaert/ZD_Putnam_Lab_Note
 - 1 uL used for D5000 Tapestation
 - 31 left over to be re-cleaned up
 
-## Results
+### Results
 
 <img src="https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/LCM_Pilot_WGBS/2024-12-16-WGBS-Cleaned-Libraries.png?raw=true" height="500">
 
 YAY! these look good but need a final clean-up to remove dimers.
 
 Full results can be found [here](https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/2024-12-16-WGBS-Cleaned-Libraries.pdf)
 
-## Final (Third) Bead cleanup to remove Dimers 
+## Third Bead cleanup to remove Dimers 
 
 - Right sided size selection protocol based on Zymo Magbead Select-a-Size clean and concentrate [protocol](https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/protocols/manual_select-a-size_dna_magbead_kit.pdf).
 
@@ -122,23 +122,38 @@ Full results can be found [here](https://github.com/zdellaert/ZD_Putnam_Lab_Note
 - 1 uL used for D5000 Tapestation
 - 30 uL final volume for Sequencing, move to LoBind tube.
 
-DONE!!! (?) Consulting with Jill if the level of dimer is okay. Still feels like I should clean again, but I am getting closer to my lower concentration limit and am worried to risk any more loss of material.
+### Results
+
+Primer dimer is still >5% of the total library. I need to clean one more time, and with a lower ratio than 0.8X.
+
+<img src="https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/LCM_Pilot_WGBS/2024-12-16-WGBS-Cleaned-Libraries-2ndclean.png?raw=true" height="500">
+
+Full results can be found [here](https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/2024-12-16-WGBS-Cleaned-Libraries-2ndclean.pdf) and [here (scaled)](https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/2024-12-16-WGBS-Cleaned-Libraries-2ndclean-scaled.pdf)
 
-<img src="https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/LCM_Pilot_WGBS/2024-12-16-WGBS-Cleaned-Libraries-cleaned.png?raw=true" height="500">
+## Final, 0.7X Bead cleanup to remove Dimers 
 
-Scaled to sample (accidentally really high contrast):
+- Right sided size selection protocol based on Zymo Magbead Select-a-Size clean and concentrate [protocol](https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/protocols/manual_select-a-size_dna_magbead_kit.pdf).
 
-<img src="https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/LCM_Pilot_WGBS/2024-12-16-WGBS-Cleaned-Libraries-cleaned-scaled.png?raw=true" height="500">
+1. Add 19 uL DNA elution buffer (EB) to bring volume to 50 uL
+2. Deplete small fragments (< 200 bp) with another volume of beads
+   1.  **0.8X ratio** = 0.7 * 50 uL = 35 uL beads
+   2. Mix thoroughly by pipetting until homogenous and incubate for 10 minutes at room temperature
+   3. Place the tube on a magnetic stand for 3 minutes, or until the supernatant is clear.
+   4. Carefully remove the supernatant without disturbing the magnetized bead pellet.
+   5. Without removing from the magnetic stand, add 200 μL of **DNA Wash Buffer** to the tube, incubate for at least 30 seconds, and then remove the supernatant completely without disturbing the magnetized bead pellet. _**Repeat this wash step**_ for two washes total.
+   6. Remove the tube from the magnetic stand and centrifuge very briefly. Then return the tube to the magnetic stand, wait for the beads to pellet, and remove any residual **DNA Wash Buffer** with a 10 μL pipette tip.
+   7. Dry on magnetic stand for 2-3 minutes with cap open, do not over-dry beads
+   8. Elute in **32 uL DNA EB**. Fully resuspend beads and incubate for 5 minutes at room temperature.
+   9. Place the tube back on the magnetic stand for 2 minutes or until the supernatant is clear.
+   10. Transfer 31 uL of eluate to a new tube. Discard the beads.
 
-Example, scaled and not scaled:
+- 1 uL used for D5000 Tapestation
+- 30 uL final volume for Sequencing, move to LoBind tube.
 
-<img src="https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/LCM_Pilot_WGBS/2024-12-16-LCM-WGBS-Trio-library-1-clean-scaled.png?raw=true" height="500">
-<img src="https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/LCM_Pilot_WGBS/2024-12-16-LCM-WGBS-Trio-library-1-clean-notscaled.png?raw=true" height="500">
+### Results
 
-But, to see the progress:
+<img src="https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/LCM_Pilot_WGBS/2024-12-17-WGBS-Final-Cleaned.png?raw=true" height="500">
 
-<img src="https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/LCM_Pilot_WGBS/2024-12-15-LCM-WGBS-Trio-library-1.png?raw=true" height="500">
-<img src="https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/LCM_Pilot_WGBS/2024-12-16-LCM-WGBS-Trio-library-1-first-clean-scaled.png?raw=true" height="500">
-<img src="https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/LCM_Pilot_WGBS/2024-12-16-LCM-WGBS-Trio-library-1-clean-scaled.png?raw=true" height="500">
+<img src="https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/LCM_Pilot_WGBS/2024-12-17-WGBS-Final-Cleaned-scaled.png?raw=true" height="500">
 
-Full results can be found [here](https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/2024-12-16-WGBS-Cleaned-Libraries-2ndclean.pdf) and [here (scaled)](https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/2024-12-16-WGBS-Cleaned-Libraries-2ndclean-scaled.pdf)
+Full results (these are the first 6 lanes, the other 4 libraries are the 12/6 and 12/12 libraries) can be found [here](https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/Final_ZD_WGBS_Trio-notscaled.pdf) and [here (scaled)](https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/images/tapestation/Final_ZD_WGBS_Trio.pdf)