Small RNA-Seq in ALKBH3 stable over expression and control HeLa cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE112870 | GEO | 2021/04/09
REPOSITORIES: GEO
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Project description:Total RNA (100 μg) were denatured at 70 °C for 5 min and separated by a 15% TBE-Urea gel with 20/100 or 10/60 oligo length standard ladder (Integrated DNA Technologies, Coralville, IA). After visualized by SYBR Gold (Ref. S11494 from Life Technologies), tRFs in gels were cut according to sizes and recovered by small RNA PAGE recovery kit (Zymo Research, Irvine, CA, USA). The recovered tRFs was used to establish the library by NEB small RNA library preparation kit (E7330S).
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Project description:Total RNA (100 μg) were denatured at 70 °C for 5 min and separated by a 15% TBE-Urea gel with 20/100 or 10/60 oligo length standard ladder (Integrated DNA Technologies, Coralville, IA). After visualized by SYBR Gold (Ref. S11494 from Life Technologies), tRFs in gels were cut according to sizes and recovered by small RNA PAGE recovery kit (Zymo Research, Irvine, CA, USA). The recovered tRFs was used to establish the library by NEB small RNA library preparation kit (E7330S).
2021-04-21 | GSE113491 | GEO
Project description:Total RNA (100 μg) were denatured at 70 °C for 5 min and separated by a 15% TBE-Urea gel with 20/100 or 10/60 oligo length standard ladder (Integrated DNA Technologies, Coralville, IA). After visualized by SYBR Gold (Ref. S11494 from Life Technologies), tRFs (15~50 nt) in gels were cut according to sizes and recovered by small RNA PAGE recovery kit (Zymo Research, Irvine, CA, USA). The recovered tRFs was used to establish the library by NEB small RNA library preparation kit (E7330S).
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Project description:The CLIP procedures were conducted using HeLa cells according to the previous study (Liu et al., 2016) with slight modifications. Briefly, 4 plates of HeLa cells in 15 cm dishes were transfected with pPB/ALKBH3 plasmid for 48 h to reach about 90% cell confluency. After washing twice with ice-cold PBS, cells were irradiated twice with 400 mJ/cm2 at 254 nm by Stratalinker on ice. Cells were lysed in high salt lysis buffer (300 mM NaCl, 0.2% NP-40, 20 mM Tris-HCl PH 7.6, 0.5 mM DTT, protease inhibitor cocktail (1 tablet/50 ml), and RNase inhibitor (1:200)) at 4°C for 30 min. Supernatant was collected after centrifuged at 17,000 g at 4°C for 15 min and further treated with 1 U RNase T1 for 15 min at 24 °C. After centrifugation and filtration through 0.22 µM filter, 10% supernatant was collected as input for further sequencing analysis. Anti-Flag M2 beads (Sigma-Aldrich, St. Louis, MO, 80 μl) were washed three times with lysis buffer and incubated with the filtered supernatant at 4°C for 4 hrs. After removing the flow through by magnetic rack, beads and input were further incubated with 10 U RNase T1 exactly for 8 min. Samples were treated with 95 µL commercial PNK buffer and 0.5 U PNK for 15 min at 37 °C. After treatments, final concentration of 100 µM ATP and 1 U PNK were added and incubated at 37 °C for another 30 min. The RNA/protein complexes were eluted by NuPAGE 1 × loading buffer and fractionated by neutral NuPAGE 4–12% bis-tris gel. After cutting the gel, RNAs were recovered by proteinase K digestion in proteinase K buffer, followed by phenol/chloroform extraction using glycogen as carrier. The RNA fragments were ligated to adapter and established library by NEB small RNA library preparation kit (E7330S).
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