Chris:LabNotes/FateMapping/Calendar/2014/2014-7-30: Difference between revisions
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>Cjwei (Created page with "=C1 DNA-seq experiment (done by Andrew and Chris)= ==Background== *We tried running the C1 DNA-seq chip for the first time using: neuN+ cortex AD patient 25-00 (sorted May 20,...") |
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==Background== | ==Background== | ||
*We tried running the C1 DNA-seq chip for the first time using: neuN+ cortex AD patient 25-00 (sorted May 20, 2014 into 1% BSA, spun down and resuspended in aliquots of 1% BSA+10% DMSO, frozen -80C) | *We tried running the C1 DNA-seq chip for the first time using: neuN+ cortex AD patient 25-00 (sorted May 20, 2014 into 1% BSA, spun down and resuspended in aliquots of 1% BSA+10% DMSO, frozen -80C) | ||
**The cell concentartion was ~256cell/ul and we aim for 250cells/ul instead of 166cells/ul in case some get lysed | |||
**Because these are neuronal nuclei, we used the small IFC | |||
*The goal of this experiment is to determine the coverage that the C1 devise provides and compare the bias to MIDAS | |||
==Procedure== | ==Procedure== | ||
===C1 DNA-seq=== | ===C1 DNA-seq=== | ||
*For most of the experiment, we used the published C1 DNA-seq protocol: [[File:C1_DNA_Seq_protocol_100-7135.pdf]] | |||
*The C1 protocol suggests to wash cells 2 times with C1 DNA Seq Cell Wash Buffer. For this run, we only washed the cells once using the following procedure: | |||
1) Add wash buffer (with 1% BSA added) to 100ul (95ul wash + 5ul sample) | |||
2) Spin down (300g 5min) | |||
3) Remove all but 5ul | |||
4) Prepare cell mix by adding 3.5ul cells + 1.5ul suspension reagent | |||
*Once the C1 finished running, utilized the PicoGreen assay as suggested by Fluidigm to quantify all the samples at once. Below is the template and results for this assay | |||
**Template: [[File:C1Dna2014Jul23PicoGreenTemplate.xlsx]] | |||
**Results: [[File:2014_07_30_results-C1Dna2014Jul23PicoGreen.xlsx]] | |||
**As seen in the above results, the concentration seems a bit lower than expected (should expect ~8-15ng/ul = 100ng output based previous discussion with Fluidigm representative) | |||
===Nextera library construction=== | ===Nextera library construction=== | ||
*We tried two methods for Nextera library construction. The first method was to use a the same procedure as done for MIDAS (treating with ALS/NS + Pol1 prior to doing Nextera). In order to account for any DNA loss during ETOH precipitation, we used 3ng for each sample. The second method was to perform library construction without Pol1 treatment in order to test whether it's necessary for the amplicons created by the C1. Because this will go directly into Nextera, we inputted only 1ng of DNA for each sample. | *We tried two methods for Nextera library construction. The first method was to use a the same procedure as done for MIDAS (treating with ALS/NS + Pol1 prior to doing Nextera). In order to account for any DNA loss during ETOH precipitation, we used 3ng for each sample. The second method was to perform library construction without Pol1 treatment in order to test whether it's necessary for the amplicons created by the C1. Because this will go directly into Nextera, we inputted only 1ng of DNA for each sample. | ||
*We also wanted to test the different results from the C1 and compare various conditions (i.e. empty vs single cell vs multiple cells). Consequently, the conditions and wells we wanted to test were: | |||
**Note: Care was taken to choose wells that were isolated (i.e. wells where the wells before/after were called empty) | |||
Condition Well | |||
call empty, <1.5ng/ul A12 | |||
call empty, >1.5ng/ul B12 | |||
call single, >1.5ng/ul G12, H12, B7 | |||
call triple, >1.5ng/ul D9 | |||
*The following is a table describing the samples and volume input from the C1 for each reaction: | *The following is a table describing the samples and volume input from the C1 for each reaction: | ||
rxn# rxn sample required mass vol input (ul) Idx | rxn# rxn sample required mass vol input (ul) Idx | ||
1 A12 (-.653ng/ul) | ----------------------------Do ALS/NS/Pol1---------------------------------- | ||
1 A12 (-.653ng/ul) 3ng 1.1895 + 0.3015 water 1 | |||
2 B12 (2.522ng/ul) | 1.1895 + 0.3015 2 | |||
3 G12 (2.614ng/ul) | 1.148 + 0.352 4 | |||
4 H12 (2.359ng/ul) | 1.272 + 0.228 5 | |||
5 B7 (1.954ng/ul) | 1.535 6 | |||
6 D9 (3.242ng/ul) | 0.925 + 0.575 7 | |||
7 gDNA (1/25d->4.4ng/ul) | 0.682 + 0.818 8 | |||
8 NTC V ---- + 1.5 10 | |||
----------------------------No ALS/NS/Pol1---------------------------------- | |||
9 A12 (do 1/10d) 1ng 3.965 + 3.035 11 | |||
10 B12 (do 1/10d) | 3.965 + 3.035 12 | |||
11 G12 (do 1/10d) | 3.826 + 3.174 13 | |||
12 H12 (do 1/10d) | 4.239 + 2.761 14 | |||
13 B7 (do 1/10d) | 5.118 + 1.882 15 | |||
14 D9 (do 1/10d) | 3.085 + 3.915 16 | |||
15 gDNA (1/250d) | 2.273 + 4.727 25 | |||
16 NTC V ---- + 7 26 | |||
==Results== | ==Results== | ||
*Below are the gels from size selection | *Below are the gels from size selection |
Revision as of 18:44, 31 July 2014
C1 DNA-seq experiment (done by Andrew and Chris)
Background
- We tried running the C1 DNA-seq chip for the first time using: neuN+ cortex AD patient 25-00 (sorted May 20, 2014 into 1% BSA, spun down and resuspended in aliquots of 1% BSA+10% DMSO, frozen -80C)
- The cell concentartion was ~256cell/ul and we aim for 250cells/ul instead of 166cells/ul in case some get lysed
- Because these are neuronal nuclei, we used the small IFC
- The goal of this experiment is to determine the coverage that the C1 devise provides and compare the bias to MIDAS
Procedure
C1 DNA-seq
- For most of the experiment, we used the published C1 DNA-seq protocol: File:C1 DNA Seq protocol 100-7135.pdf
- The C1 protocol suggests to wash cells 2 times with C1 DNA Seq Cell Wash Buffer. For this run, we only washed the cells once using the following procedure:
1) Add wash buffer (with 1% BSA added) to 100ul (95ul wash + 5ul sample) 2) Spin down (300g 5min) 3) Remove all but 5ul 4) Prepare cell mix by adding 3.5ul cells + 1.5ul suspension reagent
- Once the C1 finished running, utilized the PicoGreen assay as suggested by Fluidigm to quantify all the samples at once. Below is the template and results for this assay
- Template: File:C1Dna2014Jul23PicoGreenTemplate.xlsx
- Results: File:2014 07 30 results-C1Dna2014Jul23PicoGreen.xlsx
- As seen in the above results, the concentration seems a bit lower than expected (should expect ~8-15ng/ul = 100ng output based previous discussion with Fluidigm representative)
Nextera library construction
- We tried two methods for Nextera library construction. The first method was to use a the same procedure as done for MIDAS (treating with ALS/NS + Pol1 prior to doing Nextera). In order to account for any DNA loss during ETOH precipitation, we used 3ng for each sample. The second method was to perform library construction without Pol1 treatment in order to test whether it's necessary for the amplicons created by the C1. Because this will go directly into Nextera, we inputted only 1ng of DNA for each sample.
- We also wanted to test the different results from the C1 and compare various conditions (i.e. empty vs single cell vs multiple cells). Consequently, the conditions and wells we wanted to test were:
- Note: Care was taken to choose wells that were isolated (i.e. wells where the wells before/after were called empty)
Condition Well call empty, <1.5ng/ul A12 call empty, >1.5ng/ul B12 call single, >1.5ng/ul G12, H12, B7 call triple, >1.5ng/ul D9
- The following is a table describing the samples and volume input from the C1 for each reaction:
rxn# rxn sample required mass vol input (ul) Idx ----------------------------Do ALS/NS/Pol1---------------------------------- 1 A12 (-.653ng/ul) 3ng 1.1895 + 0.3015 water 1 2 B12 (2.522ng/ul) | 1.1895 + 0.3015 2 3 G12 (2.614ng/ul) | 1.148 + 0.352 4 4 H12 (2.359ng/ul) | 1.272 + 0.228 5 5 B7 (1.954ng/ul) | 1.535 6 6 D9 (3.242ng/ul) | 0.925 + 0.575 7 7 gDNA (1/25d->4.4ng/ul) | 0.682 + 0.818 8 8 NTC V ---- + 1.5 10 ----------------------------No ALS/NS/Pol1---------------------------------- 9 A12 (do 1/10d) 1ng 3.965 + 3.035 11 10 B12 (do 1/10d) | 3.965 + 3.035 12 11 G12 (do 1/10d) | 3.826 + 3.174 13 12 H12 (do 1/10d) | 4.239 + 2.761 14 13 B7 (do 1/10d) | 5.118 + 1.882 15 14 D9 (do 1/10d) | 3.085 + 3.915 16 15 gDNA (1/250d) | 2.273 + 4.727 25 16 NTC V ---- + 7 26
Results
- Below are the gels from size selection