Alice:Single/2015-10-23: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>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
 
(14 intermediate revisions by the same user not shown)
Line 1: Line 1:
=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)

  1. Remove the ATM, TD, and input DNA from -25°C to -15°C storage and thaw on ice
  2. Visually inspect NT to make sure that there is no precipitate. If there is precipitate, vortex until all particulates are resuspended
  3. After thawing, mix reagents by gently inverting the tubes 3–5 times, followed by a brief spin in a microcentrifuge
  4. Add 2.5 μl TD Buffer to each well to be used in this assay
  5. Add 1.25 μl diluted input DNA at 0.2 ng/μl (1 ng total) from above step
  6. Add 1.25 μl ATM to the wells containing input DNA and TD Buffer.
  7. Centrifuge at 280 × g at 20°C for 1 minute.
  8. Place in a thermal cycler and run the following program: 55°C for 5 minutes -> Hold at 10°C
  9. When the sample reaches 10°C, proceed immediately to Neutralize samples as the transposome is still active and add 1.25 μl NT Buffer
  10. vortex at medium speed briefly
  11. Centrifuge at 280 × g at 20°C for 1 minute.
  12. Leave at room temperature for 5 minutes.

PCR amplification of tagmentation treated DNA

  1. add 1.25 μl index 2 primers (white caps)
  2. add 1.25 μl index 1 primers (orange caps)
  3. add 3.75 ul NPM (amplification mix)
  4. Centrifuge at 280 × g at 20°C for 1 minute.
  5. 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: