GSM7500799: WT_RP121; Mus musculus; OTHER
HAP1 cell pellets were directly mixed with the TRIzol reagent (Invitrogen, Cat. No. 15596026), flash frozen and kept at -70°C. They were then processed according to the manufacture's protocol. To remove genomic DNA, approximately 10 µg of the extracted RNA was treated with TurboDNase (Invitrogen, Cat. No. AM2238) for 30 minutes and then cleaned by RNA Clean and Concentrator kit (Zymo Research, Cat. No. R1017). For total RNA purification from mouse livers we used multiple biological replicates (n=4) of mouse liver from control (Cmtr1loxP/+; Alb-Cre-ERT2) and Cmtr1 cKO (Cmtr1loxP/-; Alb-Cre-ERT2) animals after tamoxifen injections. Approximately 0.5 g tissue was taken and placed in a 50 mL conical tube (Sarstedt, Cat. No. 62.547.254) with 5 mL of extraction buffer. [Preparation of the extraction buffer: 250 g guanidium thiocyanate (ITW Reagents, Cat. No. A1107), 17.6 mL sodium citrate, 0.75 M, pH 7.0 (Sigma-Aldrich, Cat. No. C8532) and 320 mL water were mixed at 60°C. Add 1/10 volume of sodium acetate, 2 M, pH 4.0 (Merck, Cat. No. 1.06268 ); 1/100 volume β-mercaptoethanol (Sigma, M3148), before use.] Homogenize using a douncer (Kinematica AG, Cat. No. PT 2500E) in the 50 ml conical tube for 20 s until no fragments are left, and then add 5 mL phenol-H2O, mix well and stand on ice. Add 2 mL chloroform (VWR, Cat. No. 8.22265.2500 ): isoamyl alcohol (Merck, Cat. No. W205702 ) (49:1), mix well, stand on ice for 15 min, and transfer all the solution to a 15 mL TPP centrifuge tube (Thermo Fisher, Cat. No. 91016). Spin down for 20 min at 4000 rpm at 4°C, transfer the upper phase (approximately 5 ml) to a new 15 mL TPP tube. Then add 1 volume (~5ml) of phenol-chloroform: isoamyl alcohol (48:2) mix. Shake well to mix. Spin down for 15 min at 4000 rpm at 4°C, transfer upper phase (approximately 4 ml) to a new 15 mL TPP tube. Add 4 mL isopropanol to precipitate the nucleic acids, and leave it at −20°C for 25 min. After this, spin down at 5000rpm, 15min at 4°C, remove all the solution, and completely dry the tube by keeping it up-side down on a tissue to remove all the liquid. Add 6 mL 4M lithium chloride (Merck, Cat. No. 1.05679) to resuspend the precipitate. The volume used depends on the size of the pellet. Shake till the pellet completely dissolves. Note that the pellet contains DNA along with the RNA. Leave on ice for 5 min, and spin down at 4500 rpm for 15 min at 4°C, remove all the solution and dry the tube completely. Add 7 mL 75% ethanol to resuspend the precipitate and leave it at room temperature for 10 min. Spin down at 4500 rpm for 15 min, remove all the solution and dry the tube completely with a tissue. A pellet will be visible if the amount of RNA is abundant.. Add 75% ethanol and keep sample on ice or at -20°C for longer time. Repeat the 75% ethanol step once again. And dry the tubes at room temperature for 45min by keeping them open. Dissolve the RNA precipitation with ~700 ul DEPC treated water to a proper concentration. Dissolve RNA by gently pipetting. After a short spin, measure concentration and transfer RNA into a new 1.5ml tube. Store it under -80°C. RNA-seq library: Library preparation for embryos form the heterozygous (HET) Cmtr1+/- crosses (n=4) was performed at the Genomics Core Facility (GeneCore, EMBL Heidelberg) using 50 ng (for E6.5 embryos) and 200 ng (for E7.5 embryos) of total RNA with stranded rRNAminus RNAseq protocol and sequenced on NextSeq 500, HI, 75SE (85SE). Library preparation and sequencing of a second batch of embryos from the heterozygous (HET) Cmtr1+/- crosses or Cmtr2+/- crosses was performed at iGE3 facility at the University of Geneva. We used 50 ng (for E6.5 embryos) and 200 ng (for E7.5 embryos) of total RNA for the Smarter Ribodepletion kit. The prepared libraries were sequenced on the Illumina HiSeq 4000 sequencer (iGE3 Genomics Platform, University of Geneva). Libraries were prepared with total RNA (500 ng) from human HAP1 WT and CMTR1 KO (n=4) cells at the Genomics Core Facility (GeneCore, EMBL Heidelberg) sequencing facility using the stranded rRNAminus RNAseq protocol and sequenced on the HiSeq2000. Ribosome profiling: ribosome protected fragments (RPFs) were size-selected (25-34 nt) by electrophoresis using a 15% TBE-Urea polyacrylamide gel electrophoresis (PAGE) and two RNA markers, 25-mer (OP-RNA25) and 34-mer (OP-RNA34) (Table S1). After dephosphorylation with T4 Polynucleotide Kinase (NEB, #M0201S) the adapter Linker-1 (OP-RNA45) was ligated to the 3′ end of the RPF using T4 RNA Ligase 2 (NEB, #M0242S). The ligated products were purified using 10% TBE-Urea PAGE. Ribosomal RNA was removed using RiboCop rRNA Depletion Kit V2 H/M/R (Lexogen #144) for mouse liver samples or using the Ribo-Zero Plus rRNA Depletion kit (Illumina, #20040892) for HAP1 cell samples. The adapter Linker-1 was used for priming reverse transcription (RT) with the RT primer Ni-Ni-9 (OP27) using ProtoScript® II Reverse Transcriptase (NEB, #M0368S). RT products were purified using 10% TBE-Urea PAGE. The cDNA was circularized with CircLigase™ II ssDNA Ligase (Epicentre, #CL9021K). The final libraries were generated by PCR using forward index primer NI-N-2 (OP28) and reverse index primers. Amplified libraries were purified using 8% TBE-PAGE and analyzed with TapeStation. Libraries were sequenced on an Illumina HiSeq 4000, single-reads, 1x 50 bp, 8 libraries in a pool.