GSM5462323: HEK 293 METTL8 overexpression mitoribosome profiling; Homo sapiens; OTHER
Cells were washed once with ice cold PBS, flash frozen on liquid nitrogen and stored at -80C until lysis. 667 μL of 1.5x Lysis buffer (20 mM Tris–HCl pH 7.8, 100 mM KCl, 10 mM MgCl2, 1 mM DTT, 1% triton X-100, 0.1% NP40, 1× complete phosphatase and protease inhibitors) was added to each flask and cells were collected with a cell scraper while flasks thawed on an ice slurry. ~1 mL of lysate was transferred to a chilled 1.5 mL tube, gently passed through a 27 gauge needle 10 times, and spun at 5,000G for 10 minutes at 4C. Digestion was carried out by adding 1,500 U of MNase, 10 μL of SUPErase•In,  and CaCl2 to a final concentration of 5mM to 400 μL of lysate and incubating at 22C for 1 hour. Digestion was stopped by adding EGTA to a final concentration of 6 mM. Lysate was loaded onto a 5-30% sucrose gradient (20mM Tris–HCl pH 7.8, 100 mM KCl, 10 mM MgCl2, and 1 mM DTT and spun for 2.5 hrs at 40,000 rpm in an SW40 rotor. The 55S mitoribosome fraction was collected using a BioComp Fractionator. Footprints were isolated through SDS/hot phenol/chloroform extraction. Following each step, footprints or PCR products were cleaned by either Zymo's Oligo Clean and Concentrator or DNA Clean and Concentrator. Small RNAs were size selected using the Purelink miRNA isolation kit. 3' ends were dephosphorylated with NEB Quick CIP and 3' adapter ligation was carried out with Rnl2(1-249)K227Q ligase over night at 4°C. 3' adapter: 5'-rAppNNTGACTGTGGAATTCTCGGGTGCCAAGG-L, where the underlined sequence is the library barcode. Barcoded footprint libraries were then combined and phosphorylated by PNK. Footprints were then ligated to a 5' DNA-RNA adapter with RNA Ligase. 5'(aminolinker)GTTCAGAGTTCTACAGTCCGACGATCrNrNrNrN. Reverse transcription was carried out with SuperScript IV. RT Primer GCCTTGGCACCCGAGAATTCCA. Libraries were initially amplified by PCR using the RT primer and a short 5' primer, CTTCAGAGTTCTACAGTCCGACGA. Footprints were size selected by a Pippen Prep, 3% C gel. Sizes of 74-94 were selected, corresponding to footprint sizes of 15-35. Libraries were then amplified with long indexing primers and sequenced.