Matt:LabNotes/2013-8-16
Design 2 Probes for FISH RCA Trial[edit]
- 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[edit]
- 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 mRNA sequence: CGCCCCGCGAGCACAGAGCCTCGCCTTTGCC GAPDH: CAGCCGCATCTTCTTTTGCG'TCCTCTATGATTACTGACTGCGTCTATTTAGTGGAGCCGCTACTATCTTCTTT'TGCTCCTCCTGTTCGACAGT Matching mRNA 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
Gene | coding sequence | template DNA sequence | Left arm | Tm | Right arm | Tm |
ACTB | CGCCCCGCGAGCACAGAGCCTCGCCTTTGCC | GGCAAAGGCGAGGCTCTGTGCTCGCGGGGCG | CTGTGCTCGCGGGGCG | 67.15 | GGCAAAGGCGAGGCT | 59.97 |
RAB7A | CAGAACTTGGACCTTCTCGCTTCTGTCCTCCGTTTAGTCTCCTC | GAGGAGACTAAACGGAGGACAGAAGCGAGAAGGTCCAAGTTCTG | GAAGCGAGAAGGTCCAAGTTCTG | 63.96 | GAGGAGACTAAACGGAGGACA | 61.32 |
- coding sequence both align to RefSeq mRNA
Probe2Padlock[edit]
PadlockProbe = AP1 + LP + CommonInsert + Barcode + RP + AP2
AP1 (eMIP_CA1_F)= TGCCTAGGACCGGATCAACT (Only AGGACCGGATCAACT included in probe)
AP2 (eMIP_CA2_R)= GAGCTTCGGTTCACGCAATG (Only CATTGCGTGAACCGA included in probe)
CommonInsert (from CA12k) = CTTCAGCTTCCCGATATCCGACGG
Barcode1: dcProbe0-Cy3 = Cy3-ACGTATCGGTAGTCGCAACGCA
Barcode2: dcProbe1-Cy3 = Cy3-GTCTTGCGTGCGATACGGAGTA
ACTB: AGGACCGGATCAACT + CTGTGCTCGCGGGGCG + CTTCAGCTTCCCGATATCCGACGG + ACGTATCGGTAGTCGCAACGCA + GGCAAAGGCGAGGCT + CATTGCGTGAACCGA
RAB7A: AGGACCGGATCAACT + GAAGCGAGAAGGTCCAAGTTCTG + CTTCAGCTTCCCGATATCCGACGG + GTCTTGCGTGCGATACGGAGTA + GAGGAGACTAAACGGAGGACA + CATTGCGTGAACCGA
Full Sequence
ACTB: AGGACCGGATCAACTCTGTGCTCGCGGGGCGCTTCAGCTTCCCGATATCCGACGGACGTATCGGTAGTCGCAACGCAGGCAAAGGCGAGGCTCATTGCGTGAACCGA (107bp)
RAB7A: AGGACCGGATCAACTGAAGCGAGAAGGTCCAAGTTCTGCTTCAGCTTCCCGATATCCGACGGGTCTTGCGTGCGATACGGAGTAGAGGAGACTAAACGGAGGACACATTGCGTGAACCGA (120bp)
FINAL No Primers
- Order 100 nmole standard desalted
ppACTB: /5Phos/CTGTGCTCGCGGGGCGCTTCAGCTTCCCGATATCCGACGGACGTATCGGTAGTCGCAACGCAGGCAAAGGCGAGGCT (77bp)
ppRAB7A: /5Phos/GAAGCGAGAAGGTCCAAGTTCTGCTTCAGCTTCCCGATATCCGACGGGTCTTGCGTGCGATACGGAGTAGAGGAGACTAAACGGAGGACA (90bp)
18bp 2nd RCA Primer
"*" mark for phosphorothioate linkage
FISSEQ_ppRCA: GATATCGGGAAGCTGA*A*G Tm = 54.91