Multi-layer dosage compensation of the avian Z chromosome (bulk RNA-seq)
Multi-layer dosage compensation of the avian Z chromosome (bulk RNA-seq)
Brain, kidney, liver, skin, ovaries and testes were isolated from adult chickens. RNA isolation was performed using the phenol-chloroform extraction method. Briefly, 50-100mg of tissue was isolated and homogenised using 1 ml of TRIzol reagent [Thermofisher] and a tissue homogeniser. To precipitate the RNA, 500μl of isopropanol was added per 1 ml of TRIzol and the mixture was incubated for 10 minutes followed by centrifugation at 12000 rpm at 4°C for 10min. To wash the RNA, the pellet was resuspended in 1 ml of 75% ethanol per 1 ml of TRIzol used and centrifuged for 5 min at 7500 rpm and 4°C. The supernatant was discarded and the pellet left to air-dry for 5-10 min. The pellet was resuspended in 50μl of nuclease-free water, incubated at 55°C for 15 min and stored in -80°C.  The  concentration was measured using a Nanodrop 2000 instrument.  Cell lysis. Bulk Smart-seq2 was performed as  previously described [Larsson et al. 2019] with slight modifications. Specifically, 2ng of purified tissue RNA was added to 3μl of Smart-seq2 lysis buffer (1uM oligo-dT primer [5Biosg//idSp//idSp//idSp/ACGAGCATCAGCAGCATACGATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN; IDT], 0.5mM (each) dNTPs, 0.2% Triton-X-100, 1 U/μl RNase inhibitor [Takara]. To ensure RNA denaturation, the samples were incubated at 72°C for 3 minutes and immediately placed on ice. Reverse transcription and cDNA synthesis: 5.7 μl of reverse transcription mastermix (1x Superscript II first-strand buffer, 5mM betaine [Sigma], 6mM MgCl2 [Ambion], 1uM TSO [5'- Biotin-AGAGACAGATTGCGCAATGHHHHHHrG+GG-3'; IDT], 1.7U/μl of recombinant RNase inhibitor [Takara], 17U/μl Superscript II reverse transcriptase) was added to each sample and the reaction took place as follows: 42°C for 90min, 10 cycles of 50°C for 2 min, 42°C for 2 min, followed by, 70°C for 15 min and 4°C on hold. For the pre-amplification PCR, 15μl of PCR mastermix was added to each sample (1X Kapa HiFi HotStart ReadyMix [Roche], 0.1uM forward primers [5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGCGCAATG-3'; IDT] and 0.1uM reverse primers [5'-ACGAGCATCAGCAGCATACGA-3', IDT]) and the reaction took place as follows: 98°C for 3 min, 8 cycles of 98°C for 20 sec, 67°C for 15 sec, 72°C for 6 min, and followed by 72°C for 5 min, and 4°C on hold. cDNA purification. Purification of the cDNA libraries was performed by combining the cDNA samples to 22% PEG magnetic beads or AMPure XP beads at a ratio of 1:0.8. Briefly, the mixture was incubated at room temperature for 8 minutes and on the magnetic rack for 5 minutes. The supernatant was removed and the bead pellet was washed twice with freshly-prepared 80% ethanol. The bead pellet was left to air-dry for 3 minutes and the cDNA libraries were eluted in  17μl of EB buffer [Qiagen]. Quantification of the cDNA libraries was performed using the Quantifluor dsDNA kit [Promega]. The libraries were normalised to 1ng/ul. Tagmentation. 2ng of cDNA was combined with 18μl of tagmentation mix containing 10mM TAPS-NaOH [Sigma], 5mM MgCl2 [Thermofisher], 8% PEG-8000 and 0.5 μl of in-house produced Tn5 at 44.5uM. The samples were incubated at 55°C for 8 minutes. To strip the Tn5 off the cDNA, 3μl of 0.2% SDS solution was added to each sample and the mixture was incubated at room temperature for 5 minutes. PCR amplification. 1.5μl of combined Nextera i7 and i5 [IDT] were added to each sample as well as 25 μl of PCR mastermix (1x KAPA HiFi PCR buffer, 0.06mM (each) dNTPs, 1U KAPA HiFi polymerase). The reaction took place as follows: 72°C for 3min, 95°C for 30 sec, 10 cycles of 95°C for 10 sec, 55°C for 30 sec, 72°C for 30 sec followed by 72°C for 5 min and 4°C on hold. The libraries were pooled and purified as described above. The final concentration of the pool was measured on a Qubit 4.0 using the Qubit dsDNA High Sensitivity Assay kit [Molecular probes] and the library fragment distribution was inspected on an Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA chips. Th