Brandon:LabNotes/Project1/2015-3-4
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