title: GSM4409175: WT1; Mus musculus; ATAC-seq
library_construction_protocol: Pairs of E14.5 +/+ and SE/SE retinas (n=3 per genotype) were dissected in sterile Opti‑MEM media (Thermo Fisher) and processed into a single cell suspension by rinsing in cold PBS, and digesting extracellular matrix in TrypLE (Thermo Fisher) for 10 min at 37oC and extracellular DNA in Opti‑MEM with 0.2 U/µL DNaseI (Roche) at room temperature for 10 min.  Between steps, retinas were diluted in Opti‑MEM and centrifuged at 500×g for 5 min at 4oC.  Cells were resuspended in DMEM, dispersed by centrifugation through a pre‑wet 40 µm strainer for 2 min at 500×g at 4oC, and counted (Countess, Thermo Fisher).  Approximately 100,000 cells were washed in cold PBS, resuspended in cold cell lysis buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.3% NP-40) and immediately centrifuged for 10 min at 500×g at 4oC.  The resulting nuclear pellets were exposed to Tn5 transposase and genomic DNA (gDNA) was extracted according to the original ATAC‑seq protocol (Buenrostro et al., 2015), scaled for 100,000 cells, with the Nextera DNA Library Prep Kit (Illumina, San Diego, CA). The ATAC‑seq library was amplified using one shared primer and one unique primer, barcoded for each of six biological replicates, and half of the transposed gDNA (50,000 cell equivalents). PCR conditions and total cycle numbers were determined using previous guidelines (Buenrostro et al., 2015).  Replicates were amplified for 17, 18 and 19 cycles (wild-type) or 15, 16 and 16 cycles (SE).  Following library amplification, sample quality was assessed by polyacrylamide gel electrophoresis, Qubit DNA HS assay (Thermo Fisher) and high sensitivity DNA bioanalyzer (Agilent, Santa Clara, CA). Molar concentrations were calculated using the average fragment length and qPCR results (KAPA Library Quantification kit, Roche, Indianapolis, IN). The six sample libraries were pooled equally (300 nM each) and size-selected to remove primer and adapter dimers (SPRI beads with 1.8x ratio, Beckman Coulter, Brea, CA).  The pooled library was 1.9 ng/µL (17.5 nM) with mean fragment size ~170 bp.  This was sequenced using an Illumina HiSeq2500 instrument with a paired-end 100 format (Novogene, Sacramento, CA).
attributes.tag: GEO Accession
attributes.value: GSM4409175