title: GSM9203466: Z2KO_D0_ATAC_rep2; Mus musculus; ATAC-seq
library_construction_protocol: Assay for Transposase Accessible Chromatin (ATAC-Seq). Tn5 transposase (produced in house at EMBL Heidelberg Protein Expression and Purification Core Facility) was pre-loaded with sequencing adaptors (see Supplementary Data 4) by mixing one volume of Tn5 enzyme with half volumes of annealed R1N:ME and R2N:ME oligos (e.g., 2µl Tn5, 1µl R1N:ME, 1µl R2N:ME). The assembly reaction was incubated at room temperature for 30–60 min. ATAC-seq was performed using 20.000 nuclei per reaction. The transposition mix (containing 16 µl Transposition buffer- 38.8 mM Tris-Acetate, 77.6 mM Na-Acetate, 11.8 mM Mg-Acetate, 18.8% DMF 0.12% NP40, 0.005X Protease Inhibitor Cocktail, Nucleasefree H2O, 2 µl Assembled Tn5 + oligos and 2 µl of Nuclei suspension) was incubated at 37°C for 60 min with shaking at 500 rpm. DNA was purified using the MinElute Reaction Cleanup Kit (Qiagen, 28204), eluting in 22 µl of elution buffer. Samples were stored at -20°C prior to library preparation. To enable multiplexed sequencing, each sample (WT and Zic2-/- (Z2KO) at D0, D3, and D6) was uniquely barcoded with non-overlapping index combinations. For each sample, 20 µl of tagmented DNA were mixed with 30 µl of amplification mix and preamplified in a Veriti™ thermocycler (Applied Biosystems). To determine the optimal number of cycles, 2.5 µl of the preamplified product were subjected to RT-qPCR using 12.5 µl of qPCR mix. Based on amplification curves, the remaining PCR was completed with the minimal number of additional cycles to avoid overamplification. Amplified libraries underwent double-sided size selection using SPRIselect reagent (Beckman Coulter, B23317) at a 1.4× bead ratio, eluting in 22 µl of Elution Buffer. DNA concentrations were measured using the Qubit™ dsDNA High Sensitivity Assay Kit (Thermo Scientific, Q32854), and fragment size distribution was assessed on a 2100 Bioanalyzer using the High Sensitivity DNA Kit (Agilent, 5067-4626). Barcoded libraries were pooled and sequenced on an Illumina NextSeq2000 platform using paired-end reads and ~40 million reads per sample.