Primers for Intrabody Gene Assembly

Primers for intrabody gene assembly

Z22NLS-1 5’GAAGAAGCGGAAGGTAGGTAAGGTGCAACTTGTTGAGT 3’

Z22NLS-2 5’AGCCATGATGATGGTGATGGTGAGCTTTTGGTGCTTGT 3’

Z22NLS-3 5’GGCTAGCCACCATGGCTCCAAAGAAGAAGCGGAAGGTA 3’

Z22NLS-4 5’CTCGGATCCTTAGCCATGATGATGGTGATGGTGAGCTT 3’

Primers for assembling ZDR and control upstream reporter (NotI site)

Z1: 5’GGCCGCAAACACACACACACACACACGCGCACACACACACTCCTTACATGT 3’

Z1_REV: 5’GGCCACATGTAAGGAGTGTGTGTGTGCGCGTGTGTGTGTGTGTGTGTTTGC3’

GAPDH: 5’GGCCGCATCACCTCCCATCGGGCCACATGT 3’

GAPDH_REV: 5’GGCCACATGTGGCCCGATGGGAGGTGATGC 3’

qPCR primers for reporter gene detection.

chGAPDH forward: 5’CCATGTTCCAGGGAGATC 3’

chGAPDH reverse: 5’GCCTTCTCCATGGTGGTGAA3’

VHCD3 FORWARD:5’CCTGACGAACCCAGTGCATA3’

VHCD3 REVERSE: 5’GTTCAATCCGGTGCTGAGGT3’

Validation of ZDR

To validate ChiP sequences as ZDR we compare the Z-probe IP for the presence of the target sequences normalized for the input extract using qPCR (primers sequences below). The results were presented as fold enrichment between ΔCt values of IP of target sequences cells transfected either with Z-probe or empty vector (control), using the formulas below:

ΔCt[normalized ChIP] = (Ct[ChIP] – (Ct[Input] – log2(dilution factor)))

ΔΔCt[Z-probe/control] = ΔCt[normalized ChIPZ-probe] - ΔCt[normalized ChIPcontrol]

Fold enrichment = 2(-ΔΔCt[Z-probe/control])

qPCRprimersdesigned for detecting ZDR

ZDR / Chr. / Oligonucleotide / Sequence / %GC / σ / reference
ChIP 5.1 forward / 5’ ACC CTA GTA AGA CCA AGC CCA CTC A 3’ / 52,0
Z5.1 / 12 / ChIP 5.1 reverse / 5’ AGC GTG GTT CGG TTT GAT TTT TGT TTG 3’ / 40,7 / -0.09 / Thiswork
ChIP 7.2r forward / 5’ AGC CCC GTC TCG TGC TTA 3’ / 61,0
Z7.2 / 1 / ChIP 7.2r reverse / 5’ CTC CCG CAA CAC ACA CAA AC 3’ / 55,0 / -0.09 / Thiswork
ChIP 1.1r forward / 5’ GGG GGA CAG CAG AGCCTG GAA 3’ / 66,6
Z1.1 / 1 / ChIP 1.1r reverse / 5’ TGG TGT CTG AAC CCT GTC GTG CC 3’ / 60,8 / -0.09 / Thiswork
Chr. 4 forward / 5’ CTG TTC CTG CCC CCA CAG 3’ / 66,6 / Predictedin
Z4 / 4 / Chr. 4 reverse / 5’ CAC CTC TTT CCC ACC ACC AG 3’ / 60,0 / -0.09 / silico
CHIP 1 forward / 5’ GCG CAC ACA CAC ACT CCT TC 3’ / 60,0
Z1 / 1 / CHIP 1 reverse / 5’ GGT GCA GAC ATG ATT AGG AAA CC 3’ / 47,8 / -0.07 / Thiswork
Chr. 2 forward / 5’ GGA AAG ACG GTG CTG TGT GA 3’ / 55,0 / Predictedin
Z2 / 2 / Chr. 2 reverse / 5’ CCA GAA AGG AGGG AGGT GGT GAG T 3’ / 54,5 / -0.09 / silico
Chr. 22 forward / 5’ CTT CAT GGC AGC AGT TGG AC 3’ / 55,0
Z22 / 22 / Chr. 22 reverse / 5’ CTT CAT GGC AGC AGT TGG AC 3’ / 55,0 / -0.09 / Li et al (2009)
Chr. 19 forward / 5’ GCC AAA CGC AGA AAA GCA A 3’ / 47,3
Z19 / 19 / Chr. 19 reverse / 5’ TGA ACG CAC ACA GCA CAG AG 3’ / 55,0 / -0.09 / Li et al (2009)
Chr. X forward / 5’ GCC CCT GGA GTA GGA AGA AGA 3’ / 57,1
ZX / X / Chr. X reverse / 5’ TGA GGA GGA ATG AGC AAG CA 3’ / 50,0 / -0.09 / Li et al (2009)
hGAPDH forward / 5’ caa ttc ccc atc tca gtc gt3’ / 50,0 / Not / Control no Z
hGAPDH / 12 / hGAPDHreverse / 5’ tag tag ccg ggc cct act tt 3’ / 55,0 / found / (obtainedfrom Sigma®)

Not found – could not predict σ