Brandon:LabNotes/Project1/2015-3-4

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C1 run PCR method for accessibility on single cells with tn5-059[edit]

  • Trying again with slightly different protocol on C1. using Illumina NPM mastermix, 25 cycles of PCR, and loading mastermix into chamber 4, and backloading barcoding primers into chamber 5. both are loaded individually at 2.233X.


  • see if we can get nanograms of amplification product in the chip.


protocol[edit]

protocol summary

2. get out reagents to thaw, and cell staining mixture
3. get cells
4. load plate with harvest/blocking reagent, run priming
5. wash cells, make cell mix, and cell stain solution
6. load chip with cells etc, run cell loading and stain
7. thaw reagents for sample prep (LB, tn5 MM, protease, PCR reagents for backloading)
8. image wells on microscope
9. make solutions for (LB, tn5 MM, protease, PCR reagents for backloading)
10. load chip with LB, tn5 MM, protease, PCR reagents for backloading run sample prep script
11. get chip, remove excess barcodes from harvest wells, load harvest reagent, do harvest run, or run set to finish next morning
12. harvest samples based on how described in protocol into 96 well plate.


Cell loading (prepare during priming step):

6 uL of cells (~1400 cells total, washed in PBS)
4 uL of C1 suspension reagent

Cell staining (prepare during priming step):

1250  uL C1 cell wash buffer
2.5   uL Ethidium homodimer-1
0.625 uL calcein AM


Lysis 1.5X

15 uL 2X LB
1 uL C1LR
4 uL Nuclease free H2O


Protease 3.5X

8.750 uL protease
0.75 C1LR
5.5 uL Nuclease free H2O


Tn5 MM 2.5X

4 uL 5X D6
4 uL tn5-059 (5X)
0.4 uL C1LR


NPM only 2.233X

20.097 uL NPM
8.4 uL N-H2O
1.5 uL C1LR

i5, i7 primers 2.233X for backloading (final of 1.1165 uM)

combine C1LR and 1X TE
2180 1X TE
150 uL C1LR

for each sample in plate for i5/i7 combined
11.65 uL 1XTE/C1LR mix
1.6775 uL i5
1.6775 uL i7


  • protocol

File:Protocol Worksheet PCR Method2 v7.png




  • UPDATE 3/19/15 Exonuclease I to degrade left over primers/transposon
  • will combine 1 uL of the first 48 reactions in the plate and add Exonuclease I and buffer, before purifying with EtOH. Should not cause cross contamination issues since NPM polymerase runs at 72C.
5.5  uL 10X Exo III buffer
3.2 uL Exo I (1 uL per 20 uL)
48 uL combined samples (1 uL of each of the first 48)

37C for 15 minutes
80C for 15 minutes to inactivate
EtOH purification
1. purified with EtOH using glycoblue. Brought to 450 uL with TE. added 500 ng tRNA carrier before EtOH purfication.
2. eluted into 12.5 uL N-H2O
3. added 12.5 uL KAPA Mastermix for library quanification (from KAPA illumina library quantification kit)
   Mastmix:
    KAPA SYBR FAST
    illumina primer mix

4. ran for 10 PCR cycles, until curves reached saturation

 PCR CYCLES used:
  72°C for 3 minutes
  98°C for 30 seconds
  • 5 cycles of:
  — 98°C for 10 seconds
  — 63°C for 30 seconds
  — 72°C for 3 minutes
  • Hold at 10°C
  SAFE STOPPING POINT

5. Run on gel.


UPDATE 4/7/2015 EXOI ON ALL SAMPLES, POOLING AND PCRING

  • will add Exo I and buffer to all samples first and then incubate. after will combine 1 uL of each sample and purify with EtOH.
per barcoded sample:
1 uL (diluted to 3X buffer) 10X Exo III buffer
1 uL of 5 Unit ExoI (stock 20 Unit, dilute 4X)
3 uL sample (C1 amplicons)

37C for 30 minutes
80C for 15 minutes to inactivate

For pooling added 2.33 uL of the reactions of the first 48 (columns 1-6), and 1.8 uL of the second 48 (columns 7-12). since exoI and buffer was added, dilute the samples

EtOH purification didn't work at first, had no precipitate of glycoblue, and PCR did not work. Saved supernatent and added 2.4X volumes of EtOH reagents so it was in a total of 4 mL. Split into two 2 mL tubes and then it preciptated, and amplification was obtained with qPCR. Something with ExoIII buffer or the exoI solution did not allow it to precipitate.

EtOH purification
1. purified with EtOH using glycoblue. Brought to 450 uL with TE. added 500 ng tRNA carrier before EtOH purfication.
2. eluted into 12.5 uL N-H2O
3. added 12.5 uL KAPA Mastermix for library quanification (from KAPA illumina library quantification kit)
   Mastmix:
    KAPA SYBR FAST
    illumina primer mix

4. ran for 10 PCR cycles, until curves reached saturation

 PCR CYCLES used:
  72°C for 3 minutes
  98°C for 30 seconds
  • 5 cycles of:
  — 98°C for 10 seconds
  — 63°C for 30 seconds
  — 72°C for 3 minutes
  • Hold at 10°C
  SAFE STOPPING POINT

5. Run on gel.



UPDATE 4/21/2015, POOLING AND PCRING

  • ExoI already performed on 4/7/2015

For pooling added 4 uL 1X TE to samples 1-48 then combined and added to a 5 mL epi tube. Samples 1-48 appeared dry and thats why added 1 uL 1X TE. For samples 49-98, pipetted out the rest of the samples (~ 2 uL) into the 5 ml epi tube, then added 4 uL 1X TE to each, and pipetted that into the 5 mL epi tube.

  • used large volumes for EtOH purification since thats what found worked on 4/7/2015.
EtOH purification
1. purified with EtOH using glycoblue. Brought to 1350 uL with TE. added 1 ug tRNA carrier before EtOH purfication. 
5 mL Epi in -80C for 30 minutes, then spun down at 4500 rcf for 30 minutes. 

 volumes used
 3 mL EtOH
 120 uL NaOAc
 3 uL glycoblue

2. eluted into 12.5 uL N-H2O
3. added 12.5 uL KAPA Mastermix for library quanification (from KAPA illumina library quantification kit)
   Mastmix:
    KAPA SYBR FAST
    illumina primer mix

4. ran for 7 PCR cycles, until curves reached saturation

 PCR CYCLES used:
  95°C for 30 seconds
  • 10 cycles of:
  — 95°C for 10 seconds
  — 60°C for 30 seconds
  — 72°C for 40 seconds
  • Hold at 10°C
  SAFE STOPPING POINT

5. Run on gel.




results[edit]

Cell counts

single cells 83
2 cells 8
0 cells 5
live 66
dead 12
? 5
? Dead 1
dead dead 2
live live 5
0 cells 5


  • cells table and total counts
orig_idx well pos. capture site i5 i7 abs conc. Capture Site # of Cells live dead
17 A3 CS01 S502 N703 14.552 38.9844405 1 0
9 A2 CS02 S502 N702 17.7865 48.96903969 2 1 dead
1 A1 CS03 S502 N701 11.2125 28.67571611 3 1 live
65 A9 CS04 S502 N711 11.683 30.12810564 4 2 ? Dead
57 A8 CS05 S502 N710 15.781 42.77824863 5 1 dead
49 A7 CS06 S502 N707 12.756 33.44035636 6 1 live
18 B3 CS07 S503 N703 12.1735 31.6422333 7 1 live
10 B2 CS08 S503 N702 18.687 51.74879903 8 1 live
2 B1 CS09 S503 N701 12.1385 31.53419157 9 1 live
66 B9 CS10 S503 N711 11.4925 29.54004995 10 1 live
58 B8 CS11 S503 N710 13.836 36.77421542 11 1 live
50 B7 CS12 S503 N707 11.2915 28.91958173 12 1 live
19 C3 CS13 S505 N703 10.0485 25.0825569 13 1 live
11 C2 CS14 S505 N702 11.7325 30.28090751 14 1 live
3 C1 CS15 S505 N701 10.6905 27.0643509 15 1 live
67 C9 CS16 S505 N711 9.9545 24.79238769 16 1 live
59 C8 CS17 S505 N710 11.9815 31.04954724 17 1 ?
51 C7 CS18 S505 N707 11.737 30.29479859 18 1 live
20 D3 CS19 S506 N703 8.6035 20.62197696 19 1 live
12 D2 CS20 S506 N702 9.772 24.22902725 20 1 live
4 D1 CS21 S506 N701 13.146 34.64424991 21 1 live
68 D9 CS22 S506 N711 8.796 21.21620647 22 1 dead
60 D8 CS23 S506 N710 12.487 32.6099785 23 1 live
52 D7 CS24 S506 N707 13.3545 35.28786992 24 2 live live
5 E1 CS25 S507 N701 11.6605 30.05865024 25 1 live
13 E2 CS26 S507 N702 10.955 27.88083768 26 1 live
21 E3 CS27 S507 N703 13.2105 34.84335538 27 1 ?
53 E7 CS28 S507 N707 12.338 32.15002942 28 1 live
61 E8 CS29 S507 N710 14.5035 38.83472553 29 2 dead dead
69 E9 CS30 S507 N711 8.8355 21.33813927 30 1 live
6 F1 CS31 S508 N701 10.093 25.21992425 31 1 live
14 F2 CS32 S508 N702 10.182 25.49465893 32 2 live live
22 F3 CS33 S508 N703 7.416 16.95627544 33 1 live
54 F7 CS34 S508 N707 10.956 27.88392459 34 1 live
62 F8 CS35 S508 N710 9.4865 23.34771543 35 1 dead
70 F9 CS36 S508 N711 8.6555 20.7824961 36 1 live
7 G1 CS37 S510 N701 11.5475 29.7098298 37 1 dead
15 G2 CS38 S510 N702 9.8965 24.61334711 38 1 live
23 G3 CS39 S510 N703 11.655 30.04167226 39 1 live
55 G7 CS40 S510 N707 10.804 27.41471479 40 1 live
63 G8 CS41 S510 N710 11.0925 28.30528733 41 2 live live
71 G9 CS42 S510 N711 9.445 23.21960881 42 1 live
8 H1 CS43 S511 N701 10.7875 27.36378084 43 1 dead
16 H2 CS44 S511 N702 10.47 26.38368801 44 1 live
24 H3 CS45 S511 N703 9.857 24.4914143 45 1 live
56 H7 CS46 S511 N707 14.2595 38.08152034 46 1 live
64 H8 CS47 S511 N710 15.1575 40.85356241 47 1 live
72 H9 CS48 S511 N711 9.3015 22.77663772 48 0
25 A4 CS49 S502 N704 9.5375 23.50514766 49 1 live
33 A5 CS50 S502 N705 16.345 44.51926392 50 1 live
41 A6 CS51 S502 N706 12.822 33.64409219 51 0
73 A10 CS52 S502 N712 5.678 11.59123188 52 1 live
81 A11 CS53 S502 N714 13.202 34.81711667 53 1 live
89 A12 CS54 S502 N715 12.6375 33.07455793 54 1 live
26 B4 CS55 S503 N704 14.99 40.33650556 55 0
34 B5 CS56 S503 N705 10.43 26.26021175 56 1 live
42 B6 CS57 S503 N706 11.429 29.34403138 57 1 live
74 B10 CS58 S503 N712 8.811 21.26251006 58 1 ?
82 B11 CS59 S503 N714 10.3375 25.97467289 59 1 dead
90 B12 CS60 S503 N715 16.132 43.86175283 60 1 dead
27 C4 CS61 S505 N704 13.499 35.73392791 61 1 live
35 C5 CS62 S505 N705 11.504 29.57554937 62 1 live
43 C6 CS63 S505 N706 12.3805 32.28122295 63 1 live
75 C10 CS64 S505 N712 16.0205 43.51756275 64 1 live
83 C11 CS65 S505 N714 10.706 27.11219795 65 1 live
91 C12 CS66 S505 N715 11.0575 28.1972456 66 1 ?
28 D4 CS67 S506 N704 14.392 38.49053545 67 1 live
36 D5 CS68 S506 N705 11.794 30.47075227 68 1 live
44 D6 CS69 S506 N706 10.59 26.75411679 69 1 live
76 D10 CS70 S506 N712 12.4785 32.58373979 70 1 live
84 D11 CS71 S506 N714 12.065 31.30730394 71 1 live
92 D12 CS72 S506 N715 10.8985 27.70642746 72 1 live
45 E6 CS73 S507 N706 10.7475 27.24030457 73 2 dead dead
37 E5 CS74 S507 N705 10.01 24.963711 74 1 live
29 E4 CS75 S507 N704 12.6165 33.00973289 75 1 live
93 E12 CS76 S507 N715 14.282 38.15097573 76 2 live live
85 E11 CS77 S507 N714 10.1185 25.29864036 77 1 live
77 E10 CS78 S507 N712 11.274 28.86556087 78 1 live
46 F6 CS79 S508 N706 10.889 27.67710185 79 1 live
38 F5 CS80 S508 N705 10.86 27.58758156 80 1 ?
30 F4 CS81 S508 N704 7.7795 18.07836597 81 1 live
94 F12 CS82 S508 N715 11.649 30.02315082 82 0
86 F11 CS83 S508 N714 13.2065 34.83100775 83 2 live live
78 F10 CS84 S508 N712 12.451 32.49884986 84 1 live
47 G6 CS85 S510 N706 13.0245 34.26919076 85 1 live
39 G5 CS86 S510 N705 11.057 28.19570215 86 1 dead
31 G4 CS87 S510 N704 13.106 34.52077364 87 1 dead
95 G12 CS88 S510 N715 11.353 29.10942648 88 1 live
87 G11 CS89 S510 N714 11.076 28.25435337 89 1 live
79 G10 CS90 S510 N712 10.6665 26.99026515 90 1 live
48 H6 CS91 S511 N706 13.7845 36.61523973 91 1 live
40 H5 CS92 S511 N705 13.578 35.97779353 92 1 dead
32 H4 CS93 S511 N704 12.873 33.80152442 93 1 live
96 H12 CS94 S511 N715 11.5825 29.81787153 94 1 live
88 H11 CS95 S511 N714 12.936 33.99599953 95 1 live
80 H10 CS96 S511 N712 15.984 43.40489066 96 1 dead



3/20/15 UPDATE PREPARTION OF SEQUENCING LIBRARIES, EXOI treated

  • WORKED WELL
  • qPCR after with kapa sybr fast library quantitation mix. ran for 6 cycles after combining 1 uL of each of 48 barcodes. total of 31 cycles (25 on the chip) previously treated combined libraries with exo I then EtOH purification

File:2015-03-19 PCR 3.4.15 C1 run, pooled 1-48, 1 uL, library amplification.bmp


  • gel result after amplification (31 cycles, ExoI treated before qPCR)

File:ZhangLab 2 2015-03-19 19hr 33min-labeled.jpg


  • gel size selection

File:ZhangLab 2 2015-03-20 14hr 56min-labeled.jpg


  • validation of gel size selection

File:ZhangLab 2 2015-03-20 18hr 04min-labeled.jpg





4/7/2015 UPDATE, EXOI ON ALL INDIVIDUAL REACTIONS, POOLING, ETOH PURIFICATION THEN QPCR.

  • DIDN'T WORK TO WELL, NEEDED INCREASED VOLUME FOR ETOH PURIFICATION


  • first PCR, failed to get DNA

File:2015-04-07 PCR 3.4.15 C1 run, exoI then pooled 1-96, 1 uL, library amp.bmp


  • second PCR after increasing EtOH purification volumes 4X

File:2015-04-08 PCR 3.4.15 C1 run, exoI then pooled 1-96, 1 uL, library amp.bmp


  • after 6 cycles PCR. 1 uL for gel.

File:ZhangLab 2 2015-04-08 12hr 14min-labeled.jpg


  • gel size selection

File:ZhangLab 2 2015-04-09 13hr 34min-labeled.jpg


  • validation of gel size selection

File:ZhangLab 2 2015-04-09 16hr 30min-labeled.jpg


  • UPDATE 4/16/2015 SEQUENCING DATA ON 3/19/2015 SAMPLES, EXOI ADDED TO ALREADY POOLED SAMPLES
  • sequencing results, exoI worked, getting different mapping and clonal rates, and no overlap of the exact same reads.
sample Total Reads Mapped Reads Mapped % Clonal Reads Clonal % Total Unique Reads Total Unique % vufyd # of Cells live dead
N701-S502 28,246 7,358 26.05% 3,877 52.69% 3,481 12.32% N701-S502_TAAGGCGACTCTCTAT_L001_R1_001.fastq 1 live
N701-S503 53,248 11,925 22.40% 7,572 63.50% 4,353 8.17% N701-S503_TAAGGCGATATCCTCT_L001_R1_001.fastq 1 live
N701-S505 64,842 13,221 20.39% 8,351 63.16% 4,870 7.51% N701-S505_TAAGGCGAGTAAGGAG_L001_R1_001.fastq 1 live
N701-S506 103,105 83,551 81.03% 69,149 82.76% 14,402 13.97% N701-S506_TAAGGCGAACTGCATA_L001_R1_001.fastq 1 live
N701-S507 56,565 16,569 29.29% 11,146 67.27% 5,423 9.59% N701-S507_TAAGGCGAAAGGAGTA_L001_R1_001.fastq 1 live
N701-S508 33,833 10,050 29.70% 6,318 62.87% 3,732 11.03% N701-S508_TAAGGCGACTAAGCCT_L001_R1_001.fastq 1 live
N701-S510 48,110 22,283 46.32% 16,731 75.08% 5,552 11.54% N701-S510_TAAGGCGACGTCTAAT_L001_R1_001.fastq 1 dead
N701-S511 51,381 16,221 31.57% 11,731 72.32% 4,490 8.74% N701-S511_TAAGGCGATCTCTCCG_L001_R1_001.fastq 1 dead
N702-S502 134,502 126,740 94.23% 97,369 76.83% 29,371 21.84% N702-S502_CGTACTAGCTCTCTAT_L001_R1_001.fastq 1 dead
N702-S503 57,121 14,908 26.10% 2,399 16.09% 12,509 21.90% N702-S503_CGTACTAGTATCCTCT_L001_R1_001.fastq 1 live
N702-S505 75,515 26,663 35.31% 19,848 74.44% 6,815 9.02% N702-S505_CGTACTAGGTAAGGAG_L001_R1_001.fastq 1 live
N702-S506 76,662 32,303 42.14% 24,895 77.07% 7,408 9.66% N702-S506_CGTACTAGACTGCATA_L001_R1_001.fastq 1 live
N702-S507 92,599 38,450 41.52% 29,672 77.17% 8,778 9.48% N702-S507_CGTACTAGAAGGAGTA_L001_R1_001.fastq 1 live


  • overlap check

File:All indexs 3 4 15 overlap.png


  • comparison of accessibility regions, chr19

File:Merged wiggle comparison 3 4 15.png File:Merged wiggle comparison chr19 3 4 15.png




4/21/2015 UPDATE, EXOI ON ALL INDIVIDUAL REACTIONS (4/7/2015), POOLING, ETOH PURIFICATION in 5 mL EPI THEN QPCR, ALL 96 BARDCOES, MOSTLY LAST 48

  • WORKED WELL
  • qPCR curves. middle green lines are blanks. went down last cycle because pulled in out for last cycle then decided to do 1 more.

File:2015-04-21 3.4.15 C1 run 1 to 96 pooled, lib. amp, exoI treated indvid.png.png


  • TBE after PCR all 96 with 5 mL epi tube purification

File:ZhangLab 2 2015-04-21 16hr 05min-labeled.jpg


  • gel size selection

File:ZhangLab 2 2015-04-22 12hr 26min-labeled.jpg


  • gel size selection validation

File:ZhangLab 2 2015-04-22 16hr 35min-labeled.jpg


conclusions[edit]

  • blah