# Interdependent Transcription of a Natural Sense/Antisense Transcripts Pair (_SLC34A1_/Pfn3)
## 4 Materials and Methods
Sheared chromatin samples were diluted 1:10 (0.01% SDS, 1.1% Triton, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1 and 167 mM NaCl), protein A/G Magnetic Beads (Thermo Scientific) were added and incubated at 4 \({}^{\circ}\)C for 20 min. Pellets were washed twice with 1 mL of each low salt buffer (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), high salt buffer (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), LiCl buffer (0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1) and TE buffer (pH 8.1), followed by centrifugation (5000\(\times\) g for 1 min at 4 \({}^{\circ}\)C). Chromatin was eluted with 250 \(\upmu\)L of nuclease-free water. Reverse cross-linking was performed with proteinase K followed by phenol/chloroform extraction and ethanol precipitation. qPCR was carried out using Sybr Green I Master mix (Roche) in a LightCyclter(r) 480 System (Roche) with the following parameters: pre-incubation at 95 \({}^{\circ}\)C for 10 min; initial denaturation at 95 \({}^{\circ}\)C for 1 min; denaturation at 95 \({}^{\circ}\)C for 10 s; annealing at 57 \({}^{\circ}\)C for 30 s; and extension at 72 \({}^{\circ}\)C for 30 s for 45 cycles. All ChIP-qPCR primers are listed in Table S3. Data were normalized to rabbit anti-IgG precipitated material. The fold change (R) was determined using the formula:  
\[\text{R}=2^{\text{(Ct of H3K27 Ac/H3K4 Me3}-\text{Ct of IgG)}}\]  
CRISPR-Cas9: _SLC34A1_-sense/antisense directed sgRNAs were designed using web tools ([[https://portals.broadinstitute.org/gpp/public/analysis-tools/sgma-design](https://portals.broadinstitute.org/gpp/public/analysis-tools/sgma-design)]([https://portals.broadinstitute.org/gpp/public/analysis-tools/sgma-design](https://portals.broadinstitute.org/gpp/public/analysis-tools/sgma-design)), accessed on 24 July 2017) (Table S4). In essence, the protocol by the [PERSON] lab was followed [58]. Oligonucleotides were annealed, phosphorylated using T4 polynucleotide kinase (New England Biolabs), ligated into _BbsI_ linearized pSpCa89 (PX458) (Addgene, Watertown, MA, USA), followed by transformation of Stbl3 E. coli (Thermo Fisher). Recombinant plasmids were isolated using Plasmid Mini Kit II (Bioline, Toronto, ON, Canada), and inserts were confirmed by DNA Sanger sequencing using primer pSp-R (GATC Biotech). Transfection efficiency using HEK293 cells and Lipofectamine 2000 (Invitrogen) was tested, followed by the detection of ricked DNA to evaluate the efficiency of the different guide RNAs (EnGen Mutation Detection kit, New England Biolabs). Primers were designed to amplify a genomic region >1000 bp (Table S4). PCR products were treated with T7-endonuclease I (New England Biolabs) and separated on a 2% agarose gel, band intensity was quantified using a ChemiDoc XRS+ Imaging System (BioRad, Hercules, CA, USA). The homology repair template was designed with gene-specific arms flanking the CRISPR-Cas9 cleavage site, a transcriptional termination cassette with a puromycin antibiotic selection marker driven by the CMV promoter and a transcriptional terminator sequence, BGH-PolyA (bovine growth hormone polyadenylation) (Figure 4B). All components were amplified by PCR (primers listed in Table S5) and assembled using the NEBuilder(r) HiFi DNA Assembly Cloning Kit (New England Biolabs) at 50 \({}^{\circ}\)C overnight. Assembled fragments were examined on a 1% agarose gel, re-amplified and purified using the GeneJET PCR Purification Kit (Thermo Scientific, Waltham, MA, USA). In total, 500 ng of DNA (sgRNA containing plasmid and HDR repair template) was used to co-transfected HEK293 cells followed by cloning by serial dilution in the presence of puromycin (1 \(\upmu\)g/mL). The genomic DNA of individual clones was purified using a Wizard(r) GenomicDNA Purification Kit (Promega) followed by genotyping for integration of the HDR cassette within _SLC34A1_-sense and -antisense genomic regions.