GSM4216446: HEK293 T-Rex LARP7 ko stable FH-LARP7 F44A rescue U6 snRNA RiboMeth-Seq replicates 1-2; Homo sapiens; miRNA-Seq
Total RNA was extracted with TRIzol reagent an the RNAs of interest were purified by two consecutive pulldowns with a mix of three biotinylated antisense oligonucleotides and dynabeads M-270 streptavidin (Invitrogen). RNA samples purified by antisense oligonucleotide pulldowns were fragmented by alkaline hydrolysis in 50 mM Na2CO3 pH 9.0 for 50-55 minutes at 90°C. RNA fragments were resolved on polyacrylamide (acrylamid/bisacrylamid 19:1) urea gels, stained with SYBR gold and gel slices containing RNA fragments in the range between 21 nt and 35 nt were excised. Upon elution and precipitation of the fragments, the RNA was treated with 0.5 U/ml calf intestinal alkaline phosphatase (NEB) for 1 h at 37°C. Dephosphorylated RNAs were purified by phenol/chloroform/isoamyl alcohol extraction followed by precipitation. Phosphorylation of the 5' ends occurred with the T4 polynucleotide kinase (Thermo Scientific), followed by heat-inactivation of the enzyme, removal of unincorporated ATPs via Illustra MicroSpin G-25 gel filtration columns, phenol/chloroform/isoamyl alcohol extraction of the RNA fragments and precipitation. Small RNA libraries were generated by ligating an adenylated adapter, [5'-App-TGGAATTCTCGGGTGCCAAGG-(C7-amino)-3'], to the 3' end of the RNA fragments by a truncated T4 RNA Ligase 2. Ligated fragments were gel purified prior to ligation to the 5' RNA adapter (5'-GUUCAGAGUUCUACAGUCCGACGAUC-3') using the T4 RNA Ligase 1 (NEB). After reverse-transcription with a specific primer, the cDNA was amplified by PCR, gel-fractionated and the region above empty adapter dimers was cut out. PCR products from these gel slices were eluted in elution buffer, precipitated and dissolved in water. The quality of the libraries was assessed by measurement on a TapeStation 4200 device.
GEO Accession
GSM4216446