# Interdependent Transcription of a Natural Sense/Antisense Transcripts Pair (_SLC34A1_/Pfn3)
## 4 Materials and Methods  
Cell culture: HEK293 cells were a kind gift from [PERSON] (Newcastle) and originally purchased from Invitrogen/Thermo Fisher (Waltham, MA, USA). HKC-8 cells were obtained from Prof. [PERSON] (Newcastle) and maintained at the Cell Bank, Biosciences Institute, Newcastle University. HEK293 cells were cultured in DMEM-high glucose and HKC-8 cells in DMEM/HAM-F12 medium, supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 1% non-essential amino acids, 100 U/mL penicillin and 100 mg/mL streptomycin. Cell culture media and supplementary reagents were purchased from Sigma (St. Louis, MO, USA). Cells were seeded 24 h prior to treatment in six well plates at 0.8 \(\times\) 10\({}^{6}\) cell density. Zebularine, dexamethasone (Abcam, Cambridge, UK) and trichostatin A (Sigma) were used at various indicated concentrations. Gene expression profiling: Total RNA was extracted using a Quick-RNA Miniprep Plus Kit (Zymo Research, Orange, CA, USA) including on-column DNA digestion according to the supplier's instructions. RNA was quantified using Nano drop (Thermo Fisher, Waltham, MA, USA) and the integrity was confirmed by 2% agarose gel electrophoresis. Total RNA from kidney primary cells was obtained from Newcells (Newcastle, UK), who grow the cells from normal human kidneys according to [PERSON] et al. [57]. Human testis RNA was purchased from Takara (Kusatsu, Japan). For the RT-qPCR experiments, the Luna Universal One-Step RT-qPCR kit (New England Biolabs, Ipswich, MA, USA) was used. Samples contained 100 ng of total RNA for _SLC34A1_-sense/antisense transcripts and 25 ng for _GAPDH_. Reactions were run on a LightCyclter(r) 480 system (Roche, Basel, Switzerland) with the following parameters: Reverse transcription at 55 \({}^{\circ}\)C for 10 min; initial denaturation at 95 \({}^{\circ}\)C for 1 min; denaturation at 95 \({}^{\circ}\)C for 10 s and extension at 60 \({}^{\circ}\)C for 30 s for 45 cycles. RNA from primary renal epithelial cells and human testis RNA were included as positive controls. All primers used for the RT-qPCR and other experiments were synthesized by Integrated DNA Technologies (Coralville, IA, USA), and are listed in Tables S1-S6. The expression is given as the fold change (R) of treated vs. non-treated cells calculated as \(\Delta\Delta\)Ct = \(\Delta\)Ct (non-treated) \(-\)\(\Delta\)Ct (treated). _Pyosequencing:_ Genomic DNA was purified using the Wizard(r) Genomic DNA Purification Kit (Promega, Madison, WI, USA). In total, 450-500 ng of gDNA was subjected to sodium bisulfite treatment to deaminate un-methylated cytosines to uracils using the EZ DNA Methylation Gold Kit (Zymo Research). The promoter regions were PCR amplified using forward and biotinylated reverse primers (Table S2). PCR products were examined on a 2% agarose gel and confirmed by Sanger sequencing (GATC Biotech, Ebersberg, Germany). PCR amplicons were bound to Streptavidin Sepharose High Performance beads (GE Healthcare, Chicago, IL, USA), washed with 70% ethanol, PyroMark Denaturation Solution and PyroMark Washing buffer (Qiagen, Hilden, Germany), followed by annealing of the sequencing primers. Pyrosequencing reactions were run on a PyroMark Q96 ID (Qiagen). A positive control (100% methylation) was generated with 10 U of CpGs methyltransferase (_M.SssI_) (New England Biolabs) and unmethylated DNA (0% methylation) using a whole genome amplification kit (Qiagen). Chromatin immunoprecipitation (ChIP): Chromatin of HEK293 cells was cross-linked with 1% formaldehyde and the reaction was terminated by adding 0.125 M glycine for 5-10 min. Cell pellets were lysed in SDS lysis buffer on ice for 20 min (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1, 300 \(\upmu\)L/2-3 \(\times\) 106 cells) and sonicated using a Bioruptor(r) Pico sonicator (Diagenode, Denville, NJ, USA), with 10 cycles at high intensity, 30 s on and 20 s off.