Multi-layer dosage compensation of the avian Z chromosome (bulk RNA-seq)
Multi-layer dosage compensation of the avian Z chromosome (bulk RNA-seq)
Pellets of approximately 1 million chicken embryonic fibroblasts were pelleted at 300g for 5min and the supernatant was removed. To eliminate serum-containing media, the pellets were washed twice in 1x PBS before a final centrifugation at 300g for 5min and supernatant removal. RNA extraction was performed using Qiagen's RNeasy Plus kit according to the manufacturer's instructions. RNA concentration was measured on a Nanodrop 2000 instrument. RNA-seq library preparation was performed using Illumina's TruSeq RNA Library Prep Kit v2 according to the manufacturer's instructions.  Briefly, the samples were incubated at 65°C for 5 min, followed by bead purification to separate and elute polyA RNA-bound beads. The eluted RNA was incubated at 94°C for 8 min followed by 4°C on hold to elute, fragment and prime the RNA for first-strand synthesis. First strand synthesis was performed by adding 8 ul of first-strand master mix (containing 1ul of Superscript II reverse transcriptase for each 9 ul First Strand Master mix) to each sample and running the following program: 25°C for 10 min, 42°C for 50 min, 70°C for 15 min and hold at 4°C. For second strand synthesis, 25 ul of second strand master mix was added to each sample, followed by incubation at 16°C for 1h. After AMPure XP bead purification, end repair was performed by adding 40 ul End Repair Mix to each sample and incubating at 30°C for 30 min. After bead purification, the 3' ends were adenylated by adding 12.5 ul A-tailing mix to each sample followed by incubation at 37°C for 30 min, 70°C for 5 min and hold at 4°C. Indexing adapters were ligated by adding 2.5 ul Ligation mix and 2.5 ul RNA adapter Index (unique to each sample) per sample and the mixture was incubated at 30°C for 10 min followed by addition of 5 ul of Stop Ligation buffer to each sample to stop the ligation reaction. After bead purification, DNA fragments were enriched through PCR by adding 5 ul PCR primer cocktail and 25 ul PCR Master Mix to each sample. The reaction was performed using the following program: 98°C for 30s, 15 cycles of 98°C for 10s, 60°C for 30s, 72°C for 30s followed by 72°C for 5 min and on hold at 10°C. Finally, the libraries were  bead-purified using AMPure XP magnetic beads.  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