Chris:LabNotes/sci-Methyl Seq/Calendar/2017/2017-5-17: Difference between revisions

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(Created page with "=Test Nuclei Loss= ==Background== *We've noticed that we are getting ~75% loss of nuclei with every centrifugation during our adapter ligation. *Here are some notes from other...")
 
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=Test Nuclei Loss=
=sci-Methyl Seq Barcode 1 Design v2=
==Background==
==Background==
*We've noticed that we are getting ~75% loss of nuclei with every centrifugation during our adapter ligation.
*Previously, I've designed Adapter 1 with a CG sticky end that would anneal directly onto MspI digested DNA.  However, as previous tests have shown, this approach doesn't work because of the high amounts of adapter-adapter and template-template annealing that goes on.
*Here are some notes from other lab members about that loss:
*Dr. Zhang then suggested that we look into using end-repair/dA-tailing prior to adapter ligationThis will ensure that the majority of ligated product are template+adapterConsequently, we want to make a slight edit to the Adapter 1 test design to include a T-overhang instead of current CG overhang
**Brandon - Amount of loss was about how much he gets from working with nuclei
==Barcode 1 Adapter Design (v2)==
**Blue - normally gets only ~25% loss of nuclei with every spinSuggested that 6000xg speed centrifugation was too high and may be popping the nucleiHowever, stickiness of nuclei was something that we could not really change with centrifugation.  Also could try PBSe or 2% BSA that might help a bit
*Based on what we know from dA-tailing, an A will be added to the 3' end of blunt-ended DNAConsequently, the DNA fragment after end-repair/dA-tailing would be:
*Today, we'll be testing whether we are centrifuging too quickly, causing major sample loss.  Yesterday, Huy tried the experiment from 900xg to 6000xg and noticed that the pellet became too hard to break up >3000xg.
5'  -------------------A 3'
*Yesterday, we had some difficulty determining how to count the nucleiInitially, we gated the automated hemocytometer to 4um to 7um. However, Blue said he counts everything >4um. The reason why is because 4um can be potentially just debris and the smaller peak at 10um may either be doublets or even variability in nuclei size.
  3' A------------------- 5'
**Consequently, today, we need to figure out how to more accurately count the nuclei. One potential method would be to use the DNA content of the nuclei. This potentially may be less accurate than cell counts, but it will help us at least move past the problem of
*Consequently, our Adapter 1 v2 design should be as follows: (remember we want to create a dsDNA adapter for ligation)
==Procedure==
  5' /5Phos/------------------- 3'
*Below are the speed conditions we want to test for this experiment:
  3'      T------------------- 5'
  900xg    15min
*Below are the sequences we want to order from IDT:
  1500xg
  Adpt1_v2          5' /5Phos/TTAGAGGTGGTCCCTCCTACCCGGCGTTT 3'
  Adpt1_v2_comp    3'      TAATCTCCACCAGGGAGGATGGGCCGCAAA 5'

Latest revision as of 19:34, 17 May 2017

sci-Methyl Seq Barcode 1 Design v2[edit]

Background[edit]

  • Previously, I've designed Adapter 1 with a CG sticky end that would anneal directly onto MspI digested DNA. However, as previous tests have shown, this approach doesn't work because of the high amounts of adapter-adapter and template-template annealing that goes on.
  • Dr. Zhang then suggested that we look into using end-repair/dA-tailing prior to adapter ligation. This will ensure that the majority of ligated product are template+adapter. Consequently, we want to make a slight edit to the Adapter 1 test design to include a T-overhang instead of current CG overhang

Barcode 1 Adapter Design (v2)[edit]

  • Based on what we know from dA-tailing, an A will be added to the 3' end of blunt-ended DNA. Consequently, the DNA fragment after end-repair/dA-tailing would be:
5'  -------------------A 3'
3' A-------------------  5'
  • Consequently, our Adapter 1 v2 design should be as follows: (remember we want to create a dsDNA adapter for ligation)
5' /5Phos/------------------- 3'
3'       T------------------- 5'
  • Below are the sequences we want to order from IDT:
Adpt1_v2          5' /5Phos/TTAGAGGTGGTCCCTCCTACCCGGCGTTT 3'
Adpt1_v2_comp     3'       TAATCTCCACCAGGGAGGATGGGCCGCAAA 5'