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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)     3ng               1.1895 + 0.3015 water    1
----------------------------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

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