GSM5375182: Rpl10a_LOF_Ribo_Rep2; Mus musculus; OTHER
Whole E8.5 embryos were dissected in cold HBSS with 100 µg/mL cycloheximide and each embryo lysed for 30 minutes at 4℃ in 215 µL lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 15 mM MgCl2, 1 mM DTT, 8% glycerol, 1% Triton X-100, 100 μg/ml cycloheximide, 20 U/ml Turbo DNAse (Thermo Scientific, AM2238), and Complete Protease Inhibitor EDTA-free (Sigma-Aldrich, 11836170001)).  Lysates were sequentially centrifuged at 1300g for 5 minutes at 4℃ and then 10,000g for 10 minutes at 4℃.  70 µL of the clarified lysate was taken as the RNA input sample for RNA-seq and mixed with 55 µL water and 375 TRIzol LS (Thermo Scientific) and stored at -80C for subsequent RNA extraction.  The remaining lysate was treated with 0.5 μg RNase A (Thermo Scientific, AM2271) and 300 U RNase T1 (Thermo Scientific, EN0541) for 30 minutes at room temperature with gentle rocking. These RNases were chosen to prevent degradation of the ribosome in the small amount of material present in a single E8.5 embryo.  RNase activity was stopped by addition of 100 U SUPERase RNase Inhibitor (Thermo Scientific, AM2694). Ribosomes were enriched by sucrose cushion (1 M sucrose in lysis buffer containing 20 U/mL SUPERase RNase Inhibitor), and centrifuging in a TLA 120.2 rotor (Beckman) at 70,000 rpm for 4 hours at 4°C. The ribosome pellet containing the ribosome-protected Ribo-Seq library was resuspended in 500 μL TRIzol. Total RNA and ribosome footprints were extracted using the Direct-zol Micro Kit (Zymo) followed by DNase digest with TURBO DNase at 37℃ for 30 minutes.  DNase was removed using the RNA Clean and Concentrator 5 kit (Zymo).  The total RNA and ribosome-protected footprint samples were depleted of rRNA using Ribo-Zero Gold from the TruSeq Stranded Total RNA Library Prep Gold (Illumina 20020598) following manufacturer's instructions.  The samples were then purified with the RNA Clean & Concentrator 5 kit (Zymo) with the modifications that 2 volumes of RNA Binding Buffer and 4.5 volumes of ethanol were added to ribosome footprinting samples to purify small RNAs and 1 volume of RNA Binding Buffer and 1 volume of ethanol was added to total RNA samples to isolate RNA > 200 nt.  Total RNA samples were then diluted to 100 µL with 5 mM Tris-HCl pH 7.5, mixed with 100 µL of 2x alkaline fragmentation buffer (100 mM Na2CO3 pH 9.2, 2 mM EDTA), and incubated at 95℃ for 20 minutes to partially fragment the RNA.  The reaction was halted with 440 μL STOP Buffer (70 μL 3 M sodium acetate (NaOAc) pH 5.5, 2 μL Glycoblue, and 370 μL nuclease free water) and precipitated with isopropanol overnight at -80°C.  Fragmented total RNA and ribosome-protected footprints were size-selected by running on a 15% tris-borate-EDTA (TBE)-urea polyacrylamide gel with 20/100 ladder (IDT 51-05-15-02) to identify the 30-70 nucleotide region (for total RNA) or the oNTI199 and oNTI265 oligos to demarcate the 28-34 nucleotide region (for ribosome-protected fragments).  Gel slices were crushed and incubated at room temperature overnight in 400 μL RNA extraction buffer (300 mM NaOAc pH 5.5, 1 mM EDTA, 0.25% SDS), and the RNA precipitated with isopropanol. Samples were then 3'dephosphorylated by denaturing at 65°C for 5 minutes and incubating with 1 μL 10x T4 PNK Buffer, 1 μL SUPERase Inhibitor, 1 μL 10 U/μL T4 PNK (NEB M0201S) in a 10 µL reaction at 37°C for 1 hour followed by heat inactivation at 65°C for 20 minutes. Samples were then incubated with 0.5 μL of 50 μM Universal miRNA Cloning Linker (NEB S1315S), denatured at 65°C for 5 minutes, and mixed with 1 μL T4 RNA Ligase 2, truncated KQ (NEB M0373S), 1 μL 10x buffer, 6 μL 50% PEG 8000, and 1.5 μL water for 4.5 hours at 25°C. Free adaptor was then removed by addition of 1 μL 10 U/μL 5'-Deadenylase (NEB M0331S), 1 μL 10 U/μL RecJ Exonuclease (Lucigen/Epicentre, RJ411250), and 1 μL 20 U/μL SUPERase Inhibitor and incubation at 30℃ for 1 hour. Samples were then purified using Zymo RNA Clean and Concentrator 5 columns using the protocol above to preserve small RNAs (100 μL sample, 200 μL RNA binding buffer, 450 μL 100% ethanol).  Each 10 μL sample was then incubated with 2 μL of 1.25 μM reverse transcription primers containing sample barcodes and unique molecular identifiers and denatured at 65°C for 5 minutes, followed by reverse transcription by SuperScript III (Thermo Fisher, 18080-044) in a 20 μL reaction incubated at 25℃ for 5 minutes followed by 48°C for 40 minutes. RNA was then hydrolyzed by adding 2.2 μL of 1 N NaOH and incubating for 20 minutes at 98°C. Samples were then purified using Zymo RNA Clean & Concentrator 5 kit (100 μL sample, 200 μL RNA binding buffer, 450 μL 100% ethanol) and run on a 10% TBE-urea polyacrylamide gel. cDNA was gel extracted using DNA extraction buffer (300 mM NaCl, 10 mM Tris-HCl pH 8, 1 mM EDTA, 0.1% SDS) overnight and precipitated with isopropanol at -80°C overnight. Samples were then circularized with CircLigase (Illumina, CL4115K) in a 20 μL reaction (15 μL cDNA, 2 μL 10x CircLigase Buffer, 1 μL 1 mM ATP, 1 μL MnCl2, 1 μL CircLigase) for 12 hours at 60°C followed by a 10-minute heat inactivation at 80℃.  Samples were purified using Zymo RNA Clean & Concentrator 5 columns (100 μL sample, 200 μL RNA binding buffer, 450 μL 100% ethanol) and eluted with 12 μL 10 mM Tris-HCl pH 8. 1 μL of library was used for PCR amplification with Phusion High-Fidelity DNA Polymerase (Thermo Fisher F530S) (98°C 30 seconds, 98°C 10 seconds, 65°C 10 seconds, 72°C 5 seconds) for 10 cycles.  PCR product was run on native 8% TBE polyacrylamide gels, extracted overnight using DNA extraction buffer, and isopropanol precipitated for at least 2 hours at -80°C. Sample concentration and quality was determined by running on the Agilent 2100 Bioanalyzer (High-Sensitivity DNA) by the Stanford Protein and Nucleic Acid (PAN) Facility both before and after library pooling. Libraries were sequenced by the Stanford Functional Genomics Facility (SFGF) on the Illumina NextSeq 500 (1x75nt).