Unknown,Transcriptomics,Genomics,Proteomics

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RNA m5C Methylation in breast cancer using MeRIP-Seq

ABSTRACT: RNA m5C methylation profile of MCF10A and MDA486 by using MeRIP-Seq protocol Immunoprecipitation of Methylated mRNA at Cytosine (m5C) residues: Affinity purified of anti-methyl cytosine (m5C) polyclonal antibody 7ug (Zymo Research, Catalog#A3001-50) was conjugated with protein-A magnetic beads for 2 h at 4°C in end to end rotator. After that, conjugated beads were extensively washed with RNA immunoprecipitation (RIP) wash buffer to remove unbound antibody. Fragmented 25 ug polyA RNA (mRNA) was incubated with m5C conjugated beads for overnight at 4°C in in the rotating platform in RIP buffer. RIP was done using Megna RNA Immunoprecipitation kit (Millipore, Catalog#17-700). m5C mRNA-immune bead complex was treated with proteinase K buffer to release m5C mRNA from the conjugated antibody. To isolate m5C, mRNA was treated with phenol:chloroform:isoamyl and mixed with 400 ul of chloroform, which was centrifuged at 14000 rpm for 10 minutes to separate aqueous phase. The aqueous phase was ethanol precipitated at -80°C for overnight, to get m5C mRNA. This precipitated m5C mRNA pellet was washed twice with 70% ethanol and air dried. Finally, m5C mRNA pellet was dissolved in nuclease free Water. The m5C mRNA integrity and conentration was quantified by bioanalyzer (Agilent) and Qubit 2.0 flurometer (Invitrogen). The fragmented mRNA was used by following TruSeq RNA Sample Preparation Guide to develop RNA-Seq library for sequencing.

ORGANISM(S): Homo sapiens

SUBMITTER: Xiaodong Cui

PROVIDER: E-GEOD-53370 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Treatment of the seeds. The Solanum lycopersicum var. Money Maker wild type (MM) and fri1-1 mutant seeds were incubated on distilled-water saturated cotton wool in clear plastic boxes wrapped in black plastic sheets in darkness during 3 hours at 25°C. After that, the seeds were treated as follows: a) 24 hr of continuous irradiation with far-red light, FRc; b) 24 hr of FRc follow by a 10 min red light pulse (Rp), FRc+Rp; and c) complete darkness. After the light treatments, the seeds were incubated in darkness (25°C) until sampling. Red light (35umol/m2sec) was provided by 40W fluorescent lamps (Phillips 40/15) in combination with a red translucid acrylic. FRc light (100 umol/m2sec) was provided by a set of 150W incandescent internal reflector lamps (Phillips) in combination with a red acetate filter, six 2mm thick blue acrylic filters Paolini 2031 and 10cm water filter. Total RNA isolation. The seeds were grinded in liquid nitrogen and extracted with two volumes of extraction buffer (1% SDS, 6% p-aminosalycilic acid, 1% NaCl, 6% water-saturated phenol and 2% 2-mercaptoethanol) and two volumes of acid phenol:chloroform solution. The aqueous phase was sequentially extracted with one volume of acid phenol:chloroform solution and one volume of chloroform. The nucleic acids in the aqueous phase were precipitated with 0.15 volumes of 3M NaCl and 2.5 volumes of 100% ethanol at –80°C for 20 minutes. After centrifugation, the pellet was washed with 70% ethanol, resuspended in RNase-free water and precipitated with one volume of 4M LiCl over night at 4°C. The LiCl was removed after centrifugation and the pellet was rinsed with 2M LiCl and resuspended in RNase-free water. The total RNA was treated with RNase-free DNase (Promega) and cleaned up with mini spin columns (RNeasy Plant Mini Kit, Qiagen). The mRNA was amplificated with Message Amp II aRNA Amplification Kit (Ambion) and concentrated by vacumm centrifugation. Keywords: Direct comparison 21 hybs total

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RNA m5C Methylation in breast cancer using MeRIP-Seq

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