Chris:LabNotes/FateMapping/Calendar/2014/2014-7-30
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C1 DNA-seq experiment (done by Andrew and Chris)[edit]
Background[edit]
- 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[edit]
C1 DNA-seq[edit]
- 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[edit]
- 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 clog, >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 Sample Description ----------------------------Do ALS/NS/Pol1----------------------------------------------------------- 1 A12 (-.653ng/ul) 3ng 1.1895 + 0.3015 water 1 call empty, <1.5ng/ul 2 B12 (2.522ng/ul) | 1.1895 + 0.3015 2 call empty, >1.5ng/ul 3 G12 (2.614ng/ul) | 1.148 + 0.352 4 call single, >1.5ng/ul 4 H12 (2.359ng/ul) | 1.272 + 0.228 5 call single, >1.5ng/ul 5 B7 (1.954ng/ul) | 1.535 6 call single, >1.5ng/ul 6 D9 (3.242ng/ul) | 0.925 + 0.575 7 call clog, >1.5ng/ul 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 call empty, <1.5ng/ul 10 B12 (do 1/10d) | 3.965 + 3.035 12 call empty, >1.5ng/ul 11 G12 (do 1/10d) | 3.826 + 3.174 13 call single, >1.5ng/ul 12 H12 (do 1/10d) | 4.239 + 2.761 14 call single, >1.5ng/ul 13 B7 (do 1/10d) | 5.118 + 1.882 15 call single, >1.5ng/ul 14 D9 (do 1/10d) | 3.085 + 3.915 16 call clog, >1.5ng/ul 15 gDNA (1/250d) | 2.273 + 4.727 25 16 NTC V ---- + 7 26
- For the ALS/NS/Pol1 reactions, we want to add 1.5ul sample, so we adjusted the ALS/NS volumes accordingly to 2.25ul (consequently the Template + ALS + NS volume added into the Pol1 reaction was 6ul)
- For no ALS/NS/Pol1 reactions, we just used those directly into the Nxt transposase reactions
Results[edit]
- Below are the gels from pre-size selection
File:ZhangLab 2 2014-07-30 10hr 51min c1 pre-size selection 1.jpg
File:ZhangLab 2 2014-07-30 10hr 53min c1 pre-size selection 2.jpg
File:ZhangLab 2 2014-07-30 10hr 55min c1 pre-size selection 3.jpg
File:ZhangLab 2 2014-07-30 10hr 57min c1 pre-size selection 4.jpg
- Below are the gels from post-size selection (selected out 200-800bp)
File:ZhangLab 2 2014-07-30 11hr 39min c1 post-size selection 1.jpg File:ZhangLab 2 2014-07-30 11hr 41min c1 post-size selection 2.jpg File:ZhangLab 2 2014-07-30 11hr 42min c1 post-size selection 3.jpg File:ZhangLab 2 2014-07-30 11hr 44min c1 post-size selection 4.jpg
- Below are the usable rates after screening in HL171