Dna clean and concentrator 5 column

Standard 5’ RACE was performed exactly as described in [21 (link)]. 25% of the ligation reaction was reverse transcribed (Superscript III, Life Technologies) using the manufacturer’s gene specific primer protocol (Table S1). After RNase H treatment, cDNAs were amplified using nested PCR with the indicated (Table S1) primers. Primary nested PCR products were purified using DNA Clean and Concentrator-5 columns (Zymo). Secondary PCRs were performed using CAGE site targeting primers (Table S1) as in [21 (link)], column purified, cloned into the pGEM-T-easy vector system (Promega), transformed, plated onto plates containing ampicillin, Isopropyl β-D-1-thiogalactopyranoside (IPTG), and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal), and incubated at 37°C overnight. White colonies were assayed by colony PCR using primers complimentary to T7 and SP6 promoter sequences (Table S1). Products were separated using 2% agarose gels and ~300+ bp inserts were Sanger sequenced at the OSU Genomics Shared Resource. Sequences lacking RACE adaptors or those mapping to ribosomal RNA were discarded, and adaptor-containing sequences were identified using NCBI’s Basic Local Alignment Search Tool (BLAST) and splice variants were assigned using NCBI databases.

For each time point, 5 x 10⁵ scraped DC’s were collected by centrifugation 500 x g for 5 min. and lysed for ATAC-seq following the protocol described in (Buenrostro et al., 2015 ). Each sample was tagmented using 12.5 ul Nextera TDE-1 transposase (Illumina) for 30 minutes at 37, then quenched by addition of 5 volumes DNA Binding Buffer (Zymo Research) and cleaned using Zymo Research DNA Clean and Concentrator-5 columns according to the supplied protocol. Tagmented DNA was PCR-amplified using indexed primers as described in (Buenrostro et al., 2015 ), using total cycle numbers for enrichment as determined empirically by qPCR to minimize PCR duplicates. The resulting libraries were purified twice by Zymo Research DNA Clean and Concentrator-5 columns using a ratio of 5:1 DNA Binding Buffer:Sample, and quantified by Qubit HS-DNA Assay (Thermo Fisher Scientific) and Bioanalyzer High-Sensitivity DNA (Agilent Technologies). Final ATAC-seq libraries were pooled (equimolar) and sequenced on an Illumina Nextseq 500.

For the global chromatin accessibility experiments ductal and lobular cell lines were cultured for three days in HD conditions and then treated with 10nM estradiol for 45 minutes. Nuclei of 100,000 freshly fixed in 1% formaldehyde cells were processed as previously reported (15 (link)). Briefly, cells were resuspended in 1mL of cold ATAC-seq resuspension buffer (RSB; 10mM Tris-HCl pH 7.4, 10mM NaCl, and 3mM MgCl₂ in water) and centrifuged. Cell pellets were then resuspended in ATAC-seq RSB (0.1% NP40, 0.1% Tween-20, and 0.01% digitonin) and incubated on ice. After lysis, ATAC-seq RSB containing 0.1% Tween-20 (without NP40 or digitonin) was added. Nuclei were centrifuged and then were resuspended in 50μl of transposition mix (25μl 2× TD buffer, 2.5μl transposase (100nM final), 16.5μl PBS, 0.5μl 1% digitonin, 0.5μl 10% Tween-20, and 5μl water). Transposition reactions were incubated at 37°C for 30 min. Reactions were cleaned up with Zymo DNA Clean and Concentrator5 columns.

7 μl aliquots of all HSV target gene PCR products were analyzed for purity and specificity on a 1.8% agarose gel run in 0.5 × Tris Borate EDTA (TBE) buffer. The remaining amplified DNA was purified using DNA Clean and Concentrator-5 columns and reagents (Zymo Research) according to the manufacturer's directions. Purified PCR products were sequenced directly (ACGT, Inc) using gene specific primers. Sequences were compared to the HSV-1 genome and the NCBI database (BLASTn) to confirm specificity of each HSV target gene amplified.