title: Multi-layer dosage compensation of the avian Z chromosome (DNA-seq)
design: Multi-layer dosage compensation of the avian Z chromosome (DNA-seq)
library_construction_protocol: Chicken embryonic fibroblasts were cultured to approximately 80% confluency and pellets of 1 million cells were collected by centrifugation at 300g for 5 min. To remove as much of medium and FBS residue as possible, the pellets were washed 3 times in 1x PBS. After a final centrifugation, the supernatant was removed and pellets were placed in -80°C until DNA isolation.  Genomic DNA was extracted from frozen CEF pellets of approx 1 million cells using the Monarch Genomic DNA purification kit according to the manufacturer's instructions. Briefly, the frozen cell pellets were slowly thawed on ice and resuspended in 100 ul ice-cold PBS. 1ul Proteinase K and 3ul RNase A were added to the samples and mixed by vortexing to ensure digestion of remaining protein and RNA respectively, followed by 100ul cell lysis buffer to release the genomic DNA and incubation for 5min at 56°C with agitation at 1400 pm. To bind and elute the genomic DNA, 400ul gDNA Binding buffer to each sample and the samples were thoroughly mixed by pulse-vortexing. The lysate was then transferred to a gDNA purification column and centrifuged at 1000g for 3min and at 12000g for 1min to clear the membrane. The membrane-bound gDNA was washed twice with 500ul gDNA wash buffer by centrifuging at maximum speed for 1 min. gDNA was then eluted by adding 50ul preheated gDNA elution buffer and concentration was measured at a Nanodrop 2000 instrument. To prepare for DNA-seq library construction, gDNA was diluted to a final concentration of 1ng/ul. gDNA tagmentation was performed using an in-house prepared Tn5 enzyme as previously described (Picelli et al. 2014). Briefly, 5ng of gDNA was incubated with 15ul tagmentation master ix (10mM TAPS, 5mM MgCl2, 10% DMF, 2.25uM Tn5)at 55°C for 8min. To strip the Tn5 from the DNA, 3.5ul of freshly-prepared 0.2% SDS solution was added to each reaction and the samples were quickly centrifuged and incubated at room temperature for 5min. The samples were indexed using 2.5ul of 1uM pre-mixed Nextera index primers and post-tagmentation PCR was performed by adding 16.5 ul PCR master mix (1x KAPA HiFi PCR buffer, 0.6mM (each) dNTPs, 1U/ul KAPA HiFi polymerase) to each sample and incubating using the following program: 72°C for 3 min, 95°C for 30s, 6 cycles of [95°C for 10s, 55°C for 30s, 72°C for 30s], 72°C for 5min and 4°C on hold. Double purification of the amplified libraries was performed using in-house 22% PEG magnetic beads (Hagemann-Jensen et al. 2019).  22% PEG magnetic beads were combined with the pooled DNA libraries in a bead-to-sample ratio of 0.9:1 and incubated at room temperature for 8 minutes. The samples were then placed on a magnetic rack for 5 minutes. The clear supernatant was then removed and discarded and the bead pellets were washed twice with freshly-prepared 80% EtOH. The beads were left to air-dry for 3 minutes while remaining on the magnetic rack. The samples were eluted in 30ul. In order to ensure complete removal of residual impurities and primer-dimers, the purification was repeated as described above and the final sample was eluted in 17μl of nuclease-free water.