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>Zsakura2 (Created page with "=PGP1-iPS derived cardiomyocyte RNA sequencing= *sorted ~15,000 Tmem+VCAM1+ (day 10-12) PGP1iPS differentiated cardiomyocyte into 1.5ml microcentrifuge tube *prepared the") |
>Zsakura2 |
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=PGP1-iPS derived cardiomyocyte RNA sequencing= | =PGP1-iPS derived cardiomyocyte RNA sequencing= | ||
*sorted ~15,000 Tmem+VCAM1+ (day 10-12) PGP1iPS differentiated cardiomyocyte into 1.5ml microcentrifuge tube | *sorted ~15,000 Tmem+VCAM1+ (day 10-12) PGP1iPS differentiated cardiomyocyte into 1.5ml microcentrifuge tube | ||
*prepared the | *prepared the C1 reaction according to manufacture protocol: | ||
*Picogreen quantification | |||
{| {{table}} border=1 | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
|- | |||
| C1 well position||3||2||1||49||50||51||6||5||4||52||53||54 | |||
|- | |||
| concentration (ng/ul)||0.730||0.743||0.631||0.883||0.685||0.714||0.751||1.773||1.818||0.875||0.950||0.876 | |||
|- | |||
| C1 well position||9||8||7||55||56||57||12||11||10||58||59||60 | |||
|- | |||
| concentration (ng/ul)||1.848||1.646||0.799||0.834||0.783||0.679||1.515||1.780||1.418||1.822||0.796||0.810 | |||
|- | |||
| C1 well position||15||14||13||61||62||63||18||17||16||64||65||66 | |||
|- | |||
| concentration (ng/ul)||0.858||2.430||0.000||0.806||0.743||0.810||0.775||1.944||0.733||2.094||0.958||0.818 | |||
|- | |||
| C1 well position||21||20||19||67||68||69||24||23||22||70||71||72 | |||
|- | |||
| concentration (ng/ul)||1.833||1.461||1.841||0.825||0.751||0.816||0.857||2.390||0.814||0.865||0.888||0.881 | |||
|- | |||
| C1 well position||25||26||27||75||74||73||28||29||30||78||77||76 | |||
|- | |||
| concentration (ng/ul)||2.377||1.582||2.141||1.906||0.892||0.708||0.000||0.000||1.614||0.881||0.795||0.919 | |||
|- | |||
| C1 well position||31||32||33||81||80||79||34||35||36||84||83||82 | |||
|- | |||
| concentration (ng/ul)||2.012||3.324||0.000||0.788||0.919||0.780||0.831||0.000||0.548||0.847||0.873||0.843 | |||
|- | |||
| C1 well position||37||38||39||87||86||85||40||41||42||90||89||88 | |||
|- | |||
| concentration (ng/ul)||2.617||1.585||2.158||0.810||0.793||0.710||0.000||2.048||0.073||0.853||0.749||0.692 | |||
|- | |||
| C1 well position||43||44||45||93||92||91||46||47||48||96||95||94 | |||
|- | |||
| concentration (ng/ul)||1.750||0.863||0.669||0.773||0.853||0.795||0.000||0.853||0.344||0.658||0.658||0.658 | |||
|} | |||
*samples included for sequencing: | |||
{| {{table}} border=1 | |||
| align="center" style="background:#f0f0f0;"|'''Re-assigned #''' | |||
| align="center" style="background:#f0f0f0;"|'''96 well position''' | |||
| align="center" style="background:#f0f0f0;"|'''C1 plate position''' | |||
| align="center" style="background:#f0f0f0;"|'''Read 1 Nextera index''' | |||
| align="center" style="background:#f0f0f0;"|'''Read 2 Nextera index''' | |||
|- | |||
| 1||A9||4||S501||N701 | |||
|- | |||
| 2||A8||5||S502||N701 | |||
|- | |||
| 3||B2||8||S503||N701 | |||
|- | |||
| 4||B1||9||S504||N701 | |||
|- | |||
| 5||B9||10||S505||N701 | |||
|- | |||
| 6||B8||11||S506||N701 | |||
|- | |||
| 7||B7||12||S507||N701 | |||
|- | |||
| 8||C2||14||S508||N701 | |||
|- | |||
| 9||C8||17||S501||N702 | |||
|- | |||
| 10||D3||19||S502||N702 | |||
|- | |||
| 11||D2||20||S503||N702 | |||
|- | |||
| 12||D1||21||S504||N702 | |||
|- | |||
| 13||D8||23||S505||N702 | |||
|- | |||
| 14||E1||25||S506||N702 | |||
|- | |||
| 15||E2||26||S507||N702 | |||
|- | |||
| 16||E3||27||S508||N702 | |||
|- | |||
| 17||E9||30||S501||N703 | |||
|- | |||
| 18||F1||31||S502||N703 | |||
|- | |||
| 19||F2||32||S503||N703 | |||
|- | |||
| 20||F7?||34||S504||N703 | |||
|- | |||
| 21||G1||37||S505||N703 | |||
|- | |||
| 22||G2||38||S506||N703 | |||
|- | |||
| 23||G3||39||S507||N703 | |||
|- | |||
| 24||G8||41||S508||N704 | |||
|- | |||
| 25||H1||43||S501||N704 | |||
|- | |||
| 26||A11||53||S502||N704 | |||
|- | |||
| 27||B10||58||S503||N704 | |||
|- | |||
| 28||C10||64||S504||N704 | |||
|- | |||
| 29||E4||75||S505||N704 | |||
|} | |||
*Dilute C1 harvested samples to 0.2ng/ul according to above quantification results | |||
'''Tagmentation (Nextera XT Tagment Amplicon tube)''' | |||
#Remove the ATM, TD, and input DNA from -25°C to -15°C storage and thaw on ice | |||
#Visually inspect NT to make sure that there is no precipitate. If there is precipitate, vortex until all particulates are resuspended | |||
#After thawing, mix reagents by gently inverting the tubes 3–5 times, followed by a brief spin in a microcentrifuge | |||
#Add 2.5 μl TD Buffer to each well to be used in this assay | |||
#Add 1.25 μl diluted input DNA at 0.2 ng/μl (1 ng total) from above step | |||
#Add 1.25 μl ATM to the wells containing input DNA and TD Buffer. | |||
#Centrifuge at 280 × g at 20°C for 1 minute. | |||
#Place in a thermal cycler and run the following program: 55°C for 5 minutes -> Hold at 10°C | |||
#When the sample reaches 10°C, proceed immediately to Neutralize samples as the transposome is still active and add 1.25 μl NT Buffer | |||
#vortex at medium speed briefly | |||
#Centrifuge at 280 × g at 20°C for 1 minute. | |||
#Leave at room temperature for 5 minutes. | |||
'''PCR amplification of tagmentation treated DNA''' | |||
#add 1.25 μl index 2 primers (white caps) | |||
#add 1.25 μl index 1 primers (orange caps) | |||
#add 3.75 ul NPM (amplification mix) | |||
#Centrifuge at 280 × g at 20°C for 1 minute. | |||
#Perform PCR using the following program on a thermal cycler: | |||
*72°C for 3 minutes -> 95°C for 30 seconds -> (12 cycles of: 95°C for 10 seconds, 55°C for 30 seconds, 72°C for 30 seconds) -> 72°C for 5 minutes | |||
*pooling cDNA libraries together by taking 4ul from each well | |||
*Ampure bead XP purification twice with 0.8x concentration of bead, and elute in 50ul of H2O | |||
*gel validation of bands from 250-800bp (the smear at the far right is the sample) | |||
[[File:ZhangLab_2 2015-10-30 10hr 12min-edit.jpg]] | |||
*The sample is labeled as ZL_C1cardRNA_Oct23 and sequenced on Miseq (run folder 10302015) | |||
==Data analysis pipeline== | |||
*This pipeline uses STAR and Cufflink2. | |||
# First, copy the job submission script to your own path, such as your HOME/bin | |||
cp /home/k4zhang/bin/batch_STAR_cufflink2.pl MY_HOME/bin | |||
# Next edit this script, replace the email address "kun.zhang.ucsd@gmail.com" with my UCSD address. | |||
# This only need to be done once. | |||
*We use one script to submit jobs for all samples in one folder, one job per sample. Here is how to start the job using new version of pipeline. | |||
/home/k4zhang/bin/batch_STAR_cufflink2_HTseq.pl | |||
It handles both PE and SE reads. | |||
It uses HTSeq to report raw read counts in both exonic and intronic regions, which is more appropriate for human nuclei data. | |||
cp -r /home/k4zhang/my_oasis_tscc/test_data MY_FOLDER | |||
# For human data, replace mm10 with hg19. | |||
batch_STAR_cufflink2.pl mm10 | |||
# To check the status, use qstat. | |||
# You should also receive emails when the jobs are started or completed. | |||
qstat | grep YOUR_USER_NAME | |||
# The results are in xxx_mapped folder (one folder per sample). | |||
# To quickly get an overview of mapping stats and number of transcripts called, | |||
# transfer all xxx_mapped folders to genome-miner in a folder (GROUP_DATA_DIR) | |||
# run the following script | |||
/home/kunzhang/RNAseq/SCAP/scripts/get_STAR_mapping_stats.pl GROUP_DATA_DIR > group_name_mapping_stats.txt | |||
*Three scripts for generating summary reports on genome-miner: | |||
**[[Media: get_STAR_TPM_matrix.txt|get_STAR_TPM_matrix.pl]]: to generate TPM matrix from the raw read counts for a set of STAR mapped data. | |||
**[[Media: get_STAR_mapping_stats.txt|get_STAR_mapping_stats.pl]]: to generate a summary report on mapping statistics and ERCC correlation. | |||
**[[Media: get_STAR_geneTypes.txt|get_STAR_geneTypes.pl]]: to generate a summary report on reads mapped to different types of genes. |
Latest revision as of 00:31, 13 November 2015
PGP1-iPS derived cardiomyocyte RNA sequencing[edit]
- sorted ~15,000 Tmem+VCAM1+ (day 10-12) PGP1iPS differentiated cardiomyocyte into 1.5ml microcentrifuge tube
- prepared the C1 reaction according to manufacture protocol:
- Picogreen quantification
' | ' | ' | ' | ' | ' | ' | ' | ' | ' | ' | ' | ' |
C1 well position | 3 | 2 | 1 | 49 | 50 | 51 | 6 | 5 | 4 | 52 | 53 | 54 |
concentration (ng/ul) | 0.730 | 0.743 | 0.631 | 0.883 | 0.685 | 0.714 | 0.751 | 1.773 | 1.818 | 0.875 | 0.950 | 0.876 |
C1 well position | 9 | 8 | 7 | 55 | 56 | 57 | 12 | 11 | 10 | 58 | 59 | 60 |
concentration (ng/ul) | 1.848 | 1.646 | 0.799 | 0.834 | 0.783 | 0.679 | 1.515 | 1.780 | 1.418 | 1.822 | 0.796 | 0.810 |
C1 well position | 15 | 14 | 13 | 61 | 62 | 63 | 18 | 17 | 16 | 64 | 65 | 66 |
concentration (ng/ul) | 0.858 | 2.430 | 0.000 | 0.806 | 0.743 | 0.810 | 0.775 | 1.944 | 0.733 | 2.094 | 0.958 | 0.818 |
C1 well position | 21 | 20 | 19 | 67 | 68 | 69 | 24 | 23 | 22 | 70 | 71 | 72 |
concentration (ng/ul) | 1.833 | 1.461 | 1.841 | 0.825 | 0.751 | 0.816 | 0.857 | 2.390 | 0.814 | 0.865 | 0.888 | 0.881 |
C1 well position | 25 | 26 | 27 | 75 | 74 | 73 | 28 | 29 | 30 | 78 | 77 | 76 |
concentration (ng/ul) | 2.377 | 1.582 | 2.141 | 1.906 | 0.892 | 0.708 | 0.000 | 0.000 | 1.614 | 0.881 | 0.795 | 0.919 |
C1 well position | 31 | 32 | 33 | 81 | 80 | 79 | 34 | 35 | 36 | 84 | 83 | 82 |
concentration (ng/ul) | 2.012 | 3.324 | 0.000 | 0.788 | 0.919 | 0.780 | 0.831 | 0.000 | 0.548 | 0.847 | 0.873 | 0.843 |
C1 well position | 37 | 38 | 39 | 87 | 86 | 85 | 40 | 41 | 42 | 90 | 89 | 88 |
concentration (ng/ul) | 2.617 | 1.585 | 2.158 | 0.810 | 0.793 | 0.710 | 0.000 | 2.048 | 0.073 | 0.853 | 0.749 | 0.692 |
C1 well position | 43 | 44 | 45 | 93 | 92 | 91 | 46 | 47 | 48 | 96 | 95 | 94 |
concentration (ng/ul) | 1.750 | 0.863 | 0.669 | 0.773 | 0.853 | 0.795 | 0.000 | 0.853 | 0.344 | 0.658 | 0.658 | 0.658 |
- samples included for sequencing:
Re-assigned # | 96 well position | C1 plate position | Read 1 Nextera index | Read 2 Nextera index |
1 | A9 | 4 | S501 | N701 |
2 | A8 | 5 | S502 | N701 |
3 | B2 | 8 | S503 | N701 |
4 | B1 | 9 | S504 | N701 |
5 | B9 | 10 | S505 | N701 |
6 | B8 | 11 | S506 | N701 |
7 | B7 | 12 | S507 | N701 |
8 | C2 | 14 | S508 | N701 |
9 | C8 | 17 | S501 | N702 |
10 | D3 | 19 | S502 | N702 |
11 | D2 | 20 | S503 | N702 |
12 | D1 | 21 | S504 | N702 |
13 | D8 | 23 | S505 | N702 |
14 | E1 | 25 | S506 | N702 |
15 | E2 | 26 | S507 | N702 |
16 | E3 | 27 | S508 | N702 |
17 | E9 | 30 | S501 | N703 |
18 | F1 | 31 | S502 | N703 |
19 | F2 | 32 | S503 | N703 |
20 | F7? | 34 | S504 | N703 |
21 | G1 | 37 | S505 | N703 |
22 | G2 | 38 | S506 | N703 |
23 | G3 | 39 | S507 | N703 |
24 | G8 | 41 | S508 | N704 |
25 | H1 | 43 | S501 | N704 |
26 | A11 | 53 | S502 | N704 |
27 | B10 | 58 | S503 | N704 |
28 | C10 | 64 | S504 | N704 |
29 | E4 | 75 | S505 | N704 |
- Dilute C1 harvested samples to 0.2ng/ul according to above quantification results
Tagmentation (Nextera XT Tagment Amplicon tube)
- Remove the ATM, TD, and input DNA from -25°C to -15°C storage and thaw on ice
- Visually inspect NT to make sure that there is no precipitate. If there is precipitate, vortex until all particulates are resuspended
- After thawing, mix reagents by gently inverting the tubes 3–5 times, followed by a brief spin in a microcentrifuge
- Add 2.5 μl TD Buffer to each well to be used in this assay
- Add 1.25 μl diluted input DNA at 0.2 ng/μl (1 ng total) from above step
- Add 1.25 μl ATM to the wells containing input DNA and TD Buffer.
- Centrifuge at 280 × g at 20°C for 1 minute.
- Place in a thermal cycler and run the following program: 55°C for 5 minutes -> Hold at 10°C
- When the sample reaches 10°C, proceed immediately to Neutralize samples as the transposome is still active and add 1.25 μl NT Buffer
- vortex at medium speed briefly
- Centrifuge at 280 × g at 20°C for 1 minute.
- Leave at room temperature for 5 minutes.
PCR amplification of tagmentation treated DNA
- add 1.25 μl index 2 primers (white caps)
- add 1.25 μl index 1 primers (orange caps)
- add 3.75 ul NPM (amplification mix)
- Centrifuge at 280 × g at 20°C for 1 minute.
- Perform PCR using the following program on a thermal cycler:
- 72°C for 3 minutes -> 95°C for 30 seconds -> (12 cycles of: 95°C for 10 seconds, 55°C for 30 seconds, 72°C for 30 seconds) -> 72°C for 5 minutes
- pooling cDNA libraries together by taking 4ul from each well
- Ampure bead XP purification twice with 0.8x concentration of bead, and elute in 50ul of H2O
- gel validation of bands from 250-800bp (the smear at the far right is the sample)
File:ZhangLab 2 2015-10-30 10hr 12min-edit.jpg
- The sample is labeled as ZL_C1cardRNA_Oct23 and sequenced on Miseq (run folder 10302015)
Data analysis pipeline[edit]
- This pipeline uses STAR and Cufflink2.
# First, copy the job submission script to your own path, such as your HOME/bin cp /home/k4zhang/bin/batch_STAR_cufflink2.pl MY_HOME/bin # Next edit this script, replace the email address "kun.zhang.ucsd@gmail.com" with my UCSD address. # This only need to be done once.
- We use one script to submit jobs for all samples in one folder, one job per sample. Here is how to start the job using new version of pipeline.
/home/k4zhang/bin/batch_STAR_cufflink2_HTseq.pl It handles both PE and SE reads. It uses HTSeq to report raw read counts in both exonic and intronic regions, which is more appropriate for human nuclei data. cp -r /home/k4zhang/my_oasis_tscc/test_data MY_FOLDER # For human data, replace mm10 with hg19. batch_STAR_cufflink2.pl mm10 # To check the status, use qstat. # You should also receive emails when the jobs are started or completed. qstat | grep YOUR_USER_NAME # The results are in xxx_mapped folder (one folder per sample). # To quickly get an overview of mapping stats and number of transcripts called, # transfer all xxx_mapped folders to genome-miner in a folder (GROUP_DATA_DIR) # run the following script /home/kunzhang/RNAseq/SCAP/scripts/get_STAR_mapping_stats.pl GROUP_DATA_DIR > group_name_mapping_stats.txt
- Three scripts for generating summary reports on genome-miner:
- get_STAR_TPM_matrix.pl: to generate TPM matrix from the raw read counts for a set of STAR mapped data.
- get_STAR_mapping_stats.pl: to generate a summary report on mapping statistics and ERCC correlation.
- get_STAR_geneTypes.pl: to generate a summary report on reads mapped to different types of genes.