title: GSM6918930: NextSeq Ribo-seq, Early set, 1 dpi, rep 3; Mus musculus; OTHER
library_construction_protocol: Quick-freeze-applied lung tissue samples were homogenized within the lysis buffer (10 mM/mL Tris-HCl pH 7.4 (AM9850G, AM9855G Invitrogen), 5 mM/mL MgCl2 (#AM9530G Ambion), 100 mM/mL KCl (#AM9640G Ambion), 2 mM/mL dithiothreitol (DTT, #707265ML ThermoFisher), 300 μg/mL cycloheximide  (CHX, #C1988-1G Sigma-Aldrich), 1% Triton X-100 (#T8787 Sigma-Aldrich), 1X protease inhibitor (#P3100-001 GenDEPOT), 2 μL/mL SUPERase inhibitor (#AM2696, Invitrogen) and 2 μL/mL RNase inhibitor (#AM2694 Invitrogen)) for five minutes on ice followed by incubation at 4°C for 30 minutes with the additional lysis buffer which contained 3 times less amount of CHX. After incubation, samples were spun down and the supernatant was divided into two parts each for RNA-seq and Ribo-seq. The half for Ribo-seq was treated with 0.1 U/mL of RNase I (#EN0601 ThermoFisher) for 30 minutes at 25°C to digest the exposed mRNA from the ribosome-protected mRNA. To remove the digested mRNA fragments and collect the ribosome-protected mRNA containing lysis, samples were passed through an illustraTM MicroSpin S400 HR Columns (#27-5140-01 Cytiva) which were pre-washed with a polysome buffer (20 mM HEPES KOH pH 7.4 (#BP299100 Fisher BioReagents), 5mM MgCl2, 100 mM KCl, 2mM DTT and 100μg/mL CHX) prior to TRIzol LS treatment. Ribosomal RNAs were depleted using the RiboZero Gold Kit of TruSeq Stranded Total RNA Library Prep Gold (#20020599 Illumina) as per the manufacturer's protocol and precipitated in 100% ethanol (#100983 Supelco) at -80°C. Samples were labeled with radioisotope γ-P32 ATP (#NEG-502H-1 PerkinElmer) via T4 Polynucleotide kinase (T4 PNK, #M0201L New England Biolabs) and separated on a 10% denaturing Urea-PAGE (#U5128 Sigma-Aldrich) gel followed with signal exposure on Fujifilm imaging plates (#28956478 GE Healthcare). The radioisotope signals were visualized with an Amersham Typhoon Biomolecular imager (v2.0.0.6 GE Healthcare) and analyzed using MultiGauge (v3.0) software. Based on the separation, gel excision was performed around the ~30nt length and continued with RNA purification. Purified samples were then dephosphorylated by Antarctic Phosphatase (#M0289S New England Biolabs) and biochemically re-labeled with γ-P32 ATP using T4 PNK reaction to attach the phosphate to the free hydroxyl 5′ end of the mRNA. Additional 10mM ATP (#P0756S New England Biolabs) was supplemented to compensate for the lack of γ-P32 ATP. Samples were separated from the remaining free ATP with 10% denaturing Urea-PAGE gel followed by excision and purification, as mentioned above. Adapter ligation and PCR amplification were done by Somagenics RealSeq®-AC miRNA Library Kit (#500-00048 RealSeq Biosciences) as per the manufacturer's protocol. Desired adapter-ligated PCR products were purified through SPRIselect magnetic beads as per manufacturer's protocol, left side selection. Libraries were sequenced by the NextSeq 500 and NovaSeq 6000 systems.