Matt:LabNotes/2013-8-16

From ZhangLabWiki
Revision as of 00:46, 17 August 2013 by >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 ...")
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to navigation Jump to search

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
    • 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