title: Multi-layer dosage compensation of the avian Z chromosome (ATAC-seq)
design: Multi-layer dosage compensation of the avian Z chromosome (ATAC-seq)
library_construction_protocol: For each replicate, 100000 cells were pelleted at 500g for 5 minutes at 4°C. The supernatant was carefully removed and the pellet was resuspended in 50μl ATAC-RSB lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin) and incubated on ice for 10 min. The lysis buffer was washed out by adding 1ml of ATAC-RSB wash buffer containing 0.1% Tween but no NP-40 or digitonin and the samples inverted 3 times to mix and the nuclei pelleted at 500 RCF at 4°C for 10 min. CEFs were grown on 0.1% gelatin-coated plates in complete growth media (10% Fetal Bovine Serum [Gibco], 100U/ml  Penicillin - 100ug/ml Streptomycin, 1mM non-essential amino acids [Gibco], 1mM Sodium pyruvate [Gibco]) to 80% confluency before collection.  The supernatant was removed and the samples were resuspended in 50μl transposition mixture (25μl 2x TD buffer, 1.5μl Tn5 (27uM) 16.5 μl 1x PBS, 0.5 μl 1% digitonin, 0.5μl 10% Tween-20, 7μl water). Tagmentation was performed in a thermoshaker  at 37°C for 30 min at 1000rpm. The transposed DNA was purified using the Zymo DNA Clean and Concentrator-5 kit following the manufacturer's instructions, and eluted in 21μl of elution buffer. Library amplification was performed by adding 30μl of PCR master mix to the purified DNA (25μl 2x NEBNext High Fidelity PCR Master Mix, 2.5μl Ad1_noMX (common i5 Nextera adapter primer ), 2.5μl Ad2 (unique i7 Nextera adapter primer). The libraries were amplified for 11 cycles using the following cycling conditions: 72°C for 5 min, 98°C for 30s, 11x (98°C for 10s, 63°C for 30s, 72°C for 1 min), 4°C on hold. The final libraries were fragment size-selected by double-sided 0.5x/1.3x bead purification using homemade 22% PEG magnetic beads. Briefly, 25μl of room temperature Ampure XP beads were added to each sample (beads-to-sample ratio = 0.5) and incubated for 10 min after thorough resuspension. The samples were placed on a magnetic rack and the supernatant was removed and transferred to a new microcentrifuge tube containing 65μl room temperature Ampure XP beads (beads-to-sample (original volume) ratio = 1.3). After thorough mixing, the samples were incubated at room temperature for 10 min and placed on a magnetic rack for 5 min. The supernatant was discarded and the beads were washed twice with 200μl of freshly-prepared 80% ethanol. After ethanol removal, the samples were air-dried for 5 min and the libraries eluted in 20μl of nuclease-free water.