Multi-layer dosage compensation of the avian Z chromosome (Ribo-seq)
Multi-layer dosage compensation of the avian Z chromosome (Ribo-seq)
Cells were grown to 80 % confluency on 2x 15-cm dishes. Medium was discarded and plates were shortly submerged in liquid nitrogen to snap freeze cells. 300 μl  of 2x lysis buffer (50 mM Tris pH 7.5, 200 mM NaCl, 20 mM MgCl2, 2 mM DTT, 200 µg/ml cyclohexamide, 2 % Triton X-100, 2x Complete EDTA-free protease inhibitor cocktail (Roche), 4000 U/ml TURBO DNase I (Thermo Fisher)) was added dropwise on each plate and lysates were collected using cell scrapers. Chicken embryonic fibroblasts (CEFs) were grown on gelatin-coated 15-cm plates in DMEM supplemented with 10% heat-inactivated FBS, 1% non-essential amino acids (NEAA), 1mM sodium pyruvate, 100u/ml penicillin and 100ug/ml streptomycin. Cell debris was removed by centrifugation and RNA concentrations were measured using the Qubit RNA Broad range kit following RNAse digestion. Supernatants were loaded onto 1M sucrose cushions and ribosomes pelletted at 55 000 rpm for 3h. Supernatants were removed and the ribosomal pellets resuspended in 1ml Trizol following RNA isolation according to the manufacturer's instructions. Isolated RNA was run on a 15% Novex TBE-Urea gel and bands ranging from 25-35nt were excised, with RNA extracted from gel slices using 600 ul RNA extraction buffer  (300 mM NaOAc pH 5.5, 1 mM EDTA, 0.25 % SDS) rotating at 4 °C overnight. RNA was precipitated in ice-cold ethanol and glycoblue coprecipitant followed by pelleting and ethanol washing with subsequent resuspencion in 15ul 10 mM Tris pH 7.5. Samples were heated at 80 °C for 2 min before placing on ice. 3' phosphates were removed by T4 PNK treatment at 37 °C for 2 h. Reaction was stopped by heat inactivation (65 °C, 10 min)and RNA was pelleted by addition of 70 μl  water, 2 μl  GlycoBlue Coprecipitant (Thermo Fisher), 10 μl  1 M NaOAc and 300 μl  EtOH and subsequent storage at -80 °C. RNA was washed and dried and finally resuspended in 7 μl  10 mM Tris pH 7.5 supplemented with 1 μl  RNase inhibitor. RNA libraries were generated using TruSeq Small RNA Library Prep Kit (Illumina) according to the manufacturer's protocol with some modifications. Preparation was started by adding 1.2 μl  adenylated RA3 to dephosphorylated RNA and incubating the mixture at 80 °C for 2 min. Afterwards, ligation was performed by addition of 2 μl  of T4 RNA Ligase 2 (truncated K227Q), 2 μl  T4 RNA Ligase 2 buffer and 6 μl  PEG8000 (all components from NEB) and incubation at 14 °C ON. RNA was precipitated as described earlier, 20 μl  3 M NaOAc and 600 μl  EtOH) and resuspended in 4 μl  10 mM Tris pH 7.5. Ligation products were then purified on a 15 % Novex TBE-Urea gel (Thermo Fisher), extracted, and precipitated as described earlier. Next, RNA was resuspended in 13 μl  10 mM Tris pH 7.5 supplemented with 1 μl  RNase inhibitor. Then, 2 mM ATP, 2 μl  10x T4 PNK buffer and 2 μl  T4 PNK (NEB) were added, and the reaction mixture was incubated for 2 h at 37 °C, followed by heat inactivation (65 °C, 10 min). RNA was precipitated and resuspended in 13 μl  10 mM Tris pH 7.5 supplemented with 1 μl  RNase inhibitor. Thereafter, RNA footprints were ligated with 5' RNA adaptor (RA5, Illumina) by adding 1.2 μl  RA5, 2 μl  10x T4 buffer and 2 μl  T4 RNA ligase (Promega) and incubating at 14 °C ON. RNA was precipitated and resuspended in 3 μl  10 mM Tris pH 7.5. Reverse transcription was performed using RNA RT primers from TrueSeq Small RNA Library Prep Kit (Illumina) and SuperScript III First-Strand Synthesis System (Thermo Fisher) according to the manufacturer's protocol. Afterwards, 2 μl  of RT products were PCR amplified using Phusion High-Fidelity PCR master mix (NEB) and DNA primers from TrueSeq Small RNA Library Prep Kit (Illumina). The PCR products were resolved on a 10 % Novex non-denaturing TBE gel (Thermo Fisher) using 1x TBE running buffer. PCR products were excised and extracted using DNA extraction buffer (300 mM NaCl, 10 mM Tris pH 8, 1 mM EDTA).  Subsequently, PCR products were precipitated and pelleted. Libraries were resuspended in 12 μl  10 mM Tris pH 7.5. To reduce the amount of ribosomal RNA contamination DSN digestion was performed using a DSN kit (evrogen). First, 4 μl  of hybridization buffer (200 mM HEPES pH 7.5, 2 M NaCl) was added to the libraries. Next, libraries were heated for 2 min at 98 °C followed by incubation for 5 h at 68 °C. Consecutively, 1x master buffer (evrogen) together with 2 μl  DSN enzyme were added to the samples and incubated additional 25 min at 68 °C. Digestion was stopped by addition of 20 μl  stop solution (evrogen) and 5 min incubation at 68 °C. Finally, samples were cooled down on ice and DNA was isolated by phenol/chloroform extraction. Therefore, samples were mixed with 160 μl  water and 200 μl  phenol/chloroform (1:1) and the aqueous phase was precipitated as before. 2 μl  of digested libraries were subjected to another round of PCR amplification and consecutive gel purification. Final libraries were resuspended in 11 μl  10 mM Tris pH 7.5.