Alice:Single/2015-10-23: Difference between revisions
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*samples included for sequencing: | *samples included for sequencing: | ||
{| {{table}} | {| {{table}} border=1 | ||
| align="center" style="background:#f0f0f0;"|'''Re-assigned #''' | | align="center" style="background:#f0f0f0;"|'''Re-assigned #''' | ||
| align="center" style="background:#f0f0f0;"|'''96 well position''' | | align="center" style="background:#f0f0f0;"|'''96 well position''' | ||
| align="center" style="background:#f0f0f0;"|'''C1 plate | | align="center" style="background:#f0f0f0;"|'''C1 plate position''' | ||
| align="center" style="background:#f0f0f0;"|'''Read 1 Nextera index''' | | align="center" style="background:#f0f0f0;"|'''Read 1 Nextera index''' | ||
| align="center" style="background:#f0f0f0;"|'''Read 2 Nextera index''' | | align="center" style="background:#f0f0f0;"|'''Read 2 Nextera index''' | ||
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#Centrifuge at 280 × g at 20°C for 1 minute. | #Centrifuge at 280 × g at 20°C for 1 minute. | ||
#Perform PCR using the following program on a thermal cycler: | #Perform PCR using the following program on a thermal cycler: | ||
*72°C for 3 minutes | *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 | *pooling cDNA libraries together by taking 4ul from each well | ||
*Ampure bead XP purification with 0.8x concentration of bead, and elute in 50ul of H2O | *Ampure bead XP purification twice with 0.8x concentration of bead, and elute in 50ul of H2O | ||
*gel validation of bands from 250-800bp | *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.