Matt:LabNotes/2013-8-16

From ZhangLabWiki
Jump to navigation Jump to search

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)
 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