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=PGP1-iPS derived cardiomyocyte RNA sequencing= *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 {| {{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.
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