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===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 mRNA sequence: CGCCCCGCGAGCACAGAGCCTCGCCTTTGCC GAPDH: CAGCCGCATCTTCTTTTGCG'TCCTCTATGATTACTGACTGCGTCTATTTAGTGGAGCCGCTACTATCTTCTTT'TGCTCCTCCTGTTCGACAGT Matching mRNA sequence: TGCTCCTCCTGTTCGACAGTCAGCCGCATCTTCTTTTGCG (Using ' to separate annealing arms from rest of padlock probe sequence) *[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]] 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 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Gene''' | align="center" style="background:#f0f0f0;"|'''coding sequence''' | align="center" style="background:#f0f0f0;"|'''template DNA sequence''' | align="center" style="background:#f0f0f0;"|'''Left arm''' | align="center" style="background:#f0f0f0;"|'''Tm''' | align="center" style="background:#f0f0f0;"|'''Right arm''' | align="center" style="background:#f0f0f0;"|'''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
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