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===Design Additional Suppressor Oligos=== *Seems like suppressor oligos were not effective (enough) for the most highly counted genes **Likely due to Tm being between 55-65C and reaction temp being 55C in vitro capture or 60C for DARTFISH *Design an extra 5 suppressor oligos **Below are the top 10 probes from CA12kNov2014supp_V4gDNA_R1_H1H2_sorted_filtered_Probecounts.txt {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe (Gene_targetsequence)''' | align="center" style="background:#f0f0f0;"|'''Count''' |- | GNG4_cgacatggtgaaaccccgtctctactaaaaatacaaaaatgagcca||727150 |- | PDE1A_TTTGAacacacacacacacacacacacacacacacacacaTTCTTGTCTC||401898 |- | KCNC2_TTAATAAGAATTTATCggccgggcgcggtggctcacgcctgtaatc||156162 |- | PTPRK_tgcagtggtacaatcatagctcactgcagcctcaaattcctaggctgaag||37244 |- | GNG4_agcctggacgacagaaagagactctgtctcaaaaacaaaaacaaaaac||24799 |- | KCNN3_tccaccaccaaacccagctaatttttgtatttttagtagagatgggg||10877 |- | SLC17A7_cagcctgggcaacatagtgagaccttgtctctacaaagataaaaata||5436 |- | PPARGC1A_TACTTCCCCTAAACCAAGcacacacaccacacacatacatacacacacac||4046 |- | TESPA1_tgtgtcctcacccaaatctcatgtcaaactgtaatccccattgttggagg||3648 |} *Design options **Feature 1: Design suppressor oligos that anneal to H1 arm (Tm > 55C), has a 2bp gap, and then anneal to whole H2 arm (Tm > 68C) **Feature 2: Design suppressor oligos that complement H2 arm and continue up the barcode region ***Feature 2 makes suppressor oligos specific to V4 probeset ***Between H2 and barcode region is a poly(A) linker of variable length used make all padlock probes 150bp {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe (Gene_targetsequence)''' | align="center" style="background:#f0f0f0;"|'''Count''' | align="center" style="background:#f0f0f0;"|'''Supp Oligo''' | align="center" style="background:#f0f0f0;"|'''Left Tm''' | align="center" style="background:#f0f0f0;"|'''Right Tm''' | align="center" style="background:#f0f0f0;"|'''Old Supp Tm''' |- | GNG4_cgacatggtgaaaccccgtctctactaaaaatacaaaaatgagcca||727150||TGGTGAAACCCCGTCT AA CTACTAAAAATACAAAAATGAGCCATTTTTTTTTCTCGGTCG||58||68||57 |- | PDE1A_TTTGAacacacacacacacacacacacacacacacacacaTTCTTGTCTC||401898||ACACACACACACACAC TT ACACACACACACACATTCTTGTCTCTTTTTCTC||55||68||64 |- | KCNC2_TTAATAAGAATTTATCggccgggcgcggtggctcacgcctgtaatc||156162||GGCCGGGCGC AA GGTGGCTCACGCCTGTAATCTTTTTTTTAGC||55||69||64 |- | PTPRK_tgcagtggtacaatcatagctcactgcagcctcaaattcctaggctgaag||37244||GGTACAATCATAGCTCACTG AA CAGCCTCAAATTCCTAGGCTGAAGTTTTTCTC||56||68||64 |- | GNG4_agcctggacgacagaaagagactctgtctcaaaaacaaaaacaaaaac||24799||GGACGACAGAAAGAGACT AA CTGTCTCAAAAACAAAAACAAAAACTTTTTTTCTCGGTCG||56||68||58 |}
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