Matt:LabNotes/2013-8-16: Difference between revisions
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>Mzcai (Created page with "==Design 2 Probes for FISH RCA Trial== *Hosuk had idea to use two rounds of RCA to amplify fluorescence **First RCA is of circularized cDNA in fixed cell **The padlock probes ...") |
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*[http://www.sciencedirect.com/science/article/pii/S0168952513000899 | In Human housekeeping genes, revisited by Eisenberg, E. and Levanon, E.] they report a list (Table 1) of housekeeping genes with constant gene expression measured with RNA-Seq in Human BodyMap Project 2.0 across adrenal, adipose, brain, breast, colon, heart, kidney, liver, lung, lymph, ovary, prostate, skeletal muscle, testes, thyroid, and white blood cells | *[http://www.sciencedirect.com/science/article/pii/S0168952513000899 | In Human housekeeping genes, revisited by Eisenberg, E. and Levanon, E.] they report a list (Table 1) of housekeeping genes with constant gene expression measured with RNA-Seq in Human BodyMap Project 2.0 across adrenal, adipose, brain, breast, colon, heart, kidney, liver, lung, lymph, ovary, prostate, skeletal muscle, testes, thyroid, and white blood cells | ||
**Design probes for the mRNA transcripts in table using previous procedure: [[Matt:LabNotes/exonProbeDesign | exon probe design]] | **Design probes for the mRNA transcripts in table using previous procedure: [[Matt:LabNotes/exonProbeDesign | exon probe design]] | ||
Ensembl/Biomart all these genes except Chr1ORF43 -> mart_export_housekeeping.txt -> (add Entrez ID) -> ConstitutiveExonFinderInput_housekeeping.txt | |||
Made genes_housekeeping.txt from ncbi gene info | |||
ConstitutiveExonFinderInput_housekeeping.txt + genes_housekeeping.txt -> ConstitutiveExonFinder.pl -> TargetFile_housekeeping.txt -> TargetFileEdited_housekeepint.txt (**USED mRNA REFSEQ ACCESSION NUMBERS SO ONLY THESE TRANSCRIPTS, DIDN'T FIND OVERLAP) | |||
sortTargetFilesByChr.pl -> targetFile_chr1,3,5,9,12,15,19,22 | |||
perl src/ppDesigner.pl jobFile_chr12.pl > outputFile_chr12.txt | |||
perl src/ppDesigner.pl jobFile_chr15.pl > outputFile_chr15.txt | |||
perl src/ppDesigner.pl jobFile_chr19.pl > outputFile_chr19.txt | |||
perl src/ppDesigner.pl jobFile_chr1.pl > outputFile_chr1.txt | |||
perl src/ppDesigner.pl jobFile_chr22.pl > outputFile_chr22.txt | |||
perl src/ppDesigner.pl jobFile_chr3.pl > outputFile_chr3.txt | |||
perl src/ppDesigner.pl jobFile_chr5.pl > outputFile_chr5.txt | |||
perl src/ppDesigner.pl jobFile_chr9.pl > outputFile_chr9.txt | |||
Combined outputFiles to outputFile_housekeeping_1gap.txt | |||
ConvertToZeroGapProbe.pl -> outputFile_housekeeping_0gap.txt | |||
-> housekeeping_0gapProbes.fa | |||
**Aligned using novoalign to ref mRNA and human genome | **Aligned using novoalign to ref mRNA and human genome | ||
/home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/kunzhang/RNAseq/Data/CommonFiles/refMrna.ndx -f housekeeping_0gapProbes.fa -F FA -r ALL > housekeeping_0gapProbes_novoalign_refMrna.out | /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/kunzhang/RNAseq/Data/CommonFiles/refMrna.ndx -f housekeeping_0gapProbes.fa -F FA -r ALL > housekeeping_0gapProbes_novoalign_refMrna.out | ||
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-All 39 uniquely aligned | -All 39 uniquely aligned | ||
**Removed genes that didn't have all uniquely aligned reads, leaving: RAB7A, REEP5, VCP, and EMC7 | **Removed genes that didn't have all uniquely aligned reads, leaving: RAB7A, REEP5, VCP, and EMC7 | ||
**Took RAB7A_0 from outputFile_housekeeping_0gap_goodGenes.txt: RAB7A_0 chr3:128444990-128445177 -25 160 161 CAGAACTTGGACCTTCTCGCTTC 64 TGTCCTCCGTTTAGTCTCCTC 59 790 W CAGAACTTGGACCTTCTCGCTTCTGTCCTCCGTTTAGTCTCCTC 1.1 |
Revision as of 00:50, 17 August 2013
Design 2 Probes for FISH RCA Trial
- Hosuk had idea to use two rounds of RCA to amplify fluorescence
- First RCA is of circularized cDNA in fixed cell
- The padlock probes then capture to this rolony
- The circularized padlock probes then undergo RCA using common linker region to amplify the barcode
- Finally, fluorescent probes can be used to hybridize to the barcodes
primary RCA of cDNA -> probe capture -> secondary RCA of probes -> decoding with primers
- My task is to design two probes, one for each housekeeping gene, with two different barcodes to test the procedure
- I will try to minimize oligo length to save synthesis time/money
- 0gap padlock probe to keep as simple as possible
- Common linker region must be at least 18bp long for RCA primer
- Barcode will use one of the 22bp Cy3 SBS rolony probes in the freezer
SBS rolony probes dcProbe0-FAM FAM-ATGTATCGCGCTCGATTGGCAG dcProbe0-Cy3 Cy3-ACGTATCGGTAGTCGCAACGCA dcProbe1-FAM FAM-CAACGGTATTCGGTCGTCATCC dcProbe1-Cy3 Cy3-GTCTTGCGTGCGATACGGAGTA dcProbe2-FAM FAM-TAGAACTTGCGCGGATACACGG dcProbe2-Cy3 Cy3-TCTACTTCGTCGCGTCAGACCA dcProbe3-FAM FAM-CGAATTGTCCGCGCTCTACGAT dcProbe3-Cy3 Cy3-CCGTTTGATCGTTCGACCGAGA dcProbe4-FAM FAM-TCGGAATACGTCGTTGACTGCG dcProbe4-Cy3 Cy3-CTACCATTCGCGTGCGATTCCA
Padlock probe design: AP1 + LP + CommonLinker + Barcode + RP + AP2
- LP and RP will match (not complement) gDNA template strand because one round of RCA between cDNA and padlock capture
- Barcode will match SBS rolony probe sequence because one round of RCA between padlock circularization and probe hybridization
Picking the genes
- In 2013 NatureMethods paper Ke et al, they designed padlock probes for ACTB and GAPDH
- Picked ACTB from this because has shorter (15-16bp) annealing arms minimizing length of total oligo
ACTB: AGCCTCGCCTTTGCC'TCCTCTATGATTACTGACTGCGTCTATTTAGTGGAGCCGCATCTATCTTCTTT'CGCCCCGCGAGCACAG Matching DNA sequence: CGCCCCGCGAGCACAGAGCCTCGCCTTTGCC GAPDH: CAGCCGCATCTTCTTTTGCG'TCCTCTATGATTACTGACTGCGTCTATTTAGTGGAGCCGCTACTATCTTCTTT'TGCTCCTCCTGTTCGACAGT Matching DNA sequence: TGCTCCTCCTGTTCGACAGTCAGCCGCATCTTCTTTTGCG (Using ' to separate annealing arms from rest of padlock probe sequence)
- | In Human housekeeping genes, revisited by Eisenberg, E. and Levanon, E. they report a list (Table 1) of housekeeping genes with constant gene expression measured with RNA-Seq in Human BodyMap Project 2.0 across adrenal, adipose, brain, breast, colon, heart, kidney, liver, lung, lymph, ovary, prostate, skeletal muscle, testes, thyroid, and white blood cells
- Design probes for the mRNA transcripts in table using previous procedure: exon probe design
Ensembl/Biomart all these genes except Chr1ORF43 -> mart_export_housekeeping.txt -> (add Entrez ID) -> ConstitutiveExonFinderInput_housekeeping.txt Made genes_housekeeping.txt from ncbi gene info ConstitutiveExonFinderInput_housekeeping.txt + genes_housekeeping.txt -> ConstitutiveExonFinder.pl -> TargetFile_housekeeping.txt -> TargetFileEdited_housekeepint.txt (**USED mRNA REFSEQ ACCESSION NUMBERS SO ONLY THESE TRANSCRIPTS, DIDN'T FIND OVERLAP) sortTargetFilesByChr.pl -> targetFile_chr1,3,5,9,12,15,19,22 perl src/ppDesigner.pl jobFile_chr12.pl > outputFile_chr12.txt perl src/ppDesigner.pl jobFile_chr15.pl > outputFile_chr15.txt perl src/ppDesigner.pl jobFile_chr19.pl > outputFile_chr19.txt perl src/ppDesigner.pl jobFile_chr1.pl > outputFile_chr1.txt perl src/ppDesigner.pl jobFile_chr22.pl > outputFile_chr22.txt perl src/ppDesigner.pl jobFile_chr3.pl > outputFile_chr3.txt perl src/ppDesigner.pl jobFile_chr5.pl > outputFile_chr5.txt perl src/ppDesigner.pl jobFile_chr9.pl > outputFile_chr9.txt Combined outputFiles to outputFile_housekeeping_1gap.txt ConvertToZeroGapProbe.pl -> outputFile_housekeeping_0gap.txt -> housekeeping_0gapProbes.fa
- Aligned using novoalign to ref mRNA and human genome
/home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/kunzhang/RNAseq/Data/CommonFiles/refMrna.ndx -f housekeeping_0gapProbes.fa -F FA -r ALL > housekeeping_0gapProbes_novoalign_refMrna.out -37/39 aligned -26/37 uniquely aligned /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/kunzhang/softwares/Novocraft/novocraft/human_g1k_v37 -f housekeeping_0gapProbes.fa -F FA -r ALL > housekeeping_0gapProbes_novoalign_human_g1k_v37.out -All 39 uniquely aligned
- Removed genes that didn't have all uniquely aligned reads, leaving: RAB7A, REEP5, VCP, and EMC7
- Took RAB7A_0 from outputFile_housekeeping_0gap_goodGenes.txt: RAB7A_0 chr3:128444990-128445177 -25 160 161 CAGAACTTGGACCTTCTCGCTTC 64 TGTCCTCCGTTTAGTCTCCTC 59 790 W CAGAACTTGGACCTTCTCGCTTCTGTCCTCCGTTTAGTCTCCTC 1.1