Total RNA from PBECs or cell lines was isolated using TRIzol reagent (Thermo Fisher Scientific, Carlsbad, USA) according to the manufacturer’s protocol and RNA from LCM-collected epithelium was extracted as described above. cDNA was synthesized with random hexamer primers using the Revertaid cDNA synthesis kit (Thermo Scientific Inc.). Expression of UCHL1, collagen type I alpha 1 (COL1A1), fibronectin, collagen type III alpha 1 (COL3A1), calponin 1 (CNN1), E-cadherin (CDH1), laminin alpha 1 (LAMA1), and GAPDH genes were quantified using an ABI ViiA7 real-time PCR system (Applied Biosystems, USA) with ABsolute qPCR SYBR Green (Thermo Scientific, Inc.). Gene-specific primers are listed in Table 3 and Supplemental Table 3. Three technical replicates of real-time qRT-PCR were done for each biological repeat. Fold changes in mRNA expression above the control (dCas9-NED transfected cells) were calculated by the cycle threshold (ΔΔCt) method after normalization to GAPDH expression, unless stated otherwise.

Information of PCR and sequencing primers.

PrimerSequence (5’-3’)Application
UCHL1-FwTTCCTGTGGCACAATCGGACqRT-PCR primer for UCHL1
UCHL1-RvCATCTACCCGACATTGGCCTT
COL1A1-FwGGGATTCCCTGGACCTAAAGqRT-PCR primer for COL1A1
COL1A1-RvGGAACACCTCGCTCTCCA
COL3A1-FwCTGGACCCCAGGGTCTTCqRT-PCR primer for COL3A1
COL3A1-RvCATCTGATCCAGGGTTTCCA
Fibronectin-FwTCAACTCACAGCTTCTCCAAqRT-PCR primer for Fibronectin
Fibronectin-RvTTGATCCCAAACCAAATCTT
CNN1-FwCCAACCATACACAGGTGCAGqRT-PCR primer for CNN1
CNN1-RvTCACCTTGTTTCCTTTCGTCTT
CDH1-FwCATTGCCACATACACTCTCTTCTqRT-PCR primer for CDH1
CDH1-RvCGGTTACCGTGATCAAAATCTC
GAPDH-FwCCACATCGCTCAGACACCATqRT-PCR primer for GAPDH
GAPDH-RvGCGCCCAATACGACCAAAT
dCas9-FwAATGGCATCCGAGACAAGCAqRT-PCR primer for dCas9
dCas9-RvTGTGCTCGTGAAGACTGTCC
UCHL1-pyro-FwGGTTTTGTTTTTGTTTTTTTTGTATAGGPCR and sequencing primer for UCHL1 pyrosequencing (lower cases reflect the universal primer, Y is the CpG sites tested, subscript number indicating the site) (Site #4 is SNP-rs577696101-C/G according to UCSC)
UCHL1-pyro-RvgggacaccgctgatcgtttaAATCTCCA-TCYACTTAAACTACATCTTC
Pyroseq-sequencing primerTTGTATAGGTTTTATAGTG
Pyroseq-sequence to analyseY1GTTTGGTY2GGY3GTTTTATAGTTGTAGTTTGGGY4GGTTTY5G TTAGTTGTTTTTY6GTTTTTTTTAGGTTATTTTTGTY7GGGYGTTTYGYGAAGATGTAGTTTAAGTYGATGGAGATT
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