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Old 04-04-2016, 10:22 AM   #5
ismail selim
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Location: Turkey

Join Date: Mar 2016
Posts: 2
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I starting with 119 ng/Ál broad rage RNA kit, use for ribosomal RNA removing NEbnext depletion kit, dynabead for mRNA removing, concentrate with using RNA clean &Concentrator Zymo. Than fragmentation with Mg++2 (Nebnext fragmentation kit) for more efficient effecitive data representing 3' and 5' ends. The sample were incubated at 84◦C, 7 min. Following fragmentation the terminal ends of the RNA wereprepared for adaptor ligation. Antarctic Phosphatase (New EnglandBiolabs, Ipswich, MA, USA).Next, samples were treated with Polynu-cleotide Kinase (New England Biolabs, Ipswich, MA, USA) to add a 5phosphate, according to the manufacturer’s protocol. FragmentedRNA was cleaned up again with the RNeasy MinElute Spin ColumnKit (Qiagen, Germantown, MD, USA) according to the manufac-turer’s protocol.Adapter ligation was performed in two steps usingreagents from Illumina’s TruSeq Small RNA Sample Preparation kit(Illumina, San Diego, CA, USA). First, the RNA 3Adapter T4 RNA ligase 2, truncated (New England Biolabs, Ipswich,MA, USA.Next, the 5adapter was ligated to theRNA T4 RNA ligase wereadded to the RNA 5adapter solution.PCR enrichment, usingIllumina’s TruSeq Small RNA Sample Prep Kit protocol and reagents(Illumina, San Diego, CA, USA). Lastly, the samples underwent twofinal cleanups with Agencourt AMpure XP Beads (Beckman Coul-ter, Irving, TX, USA) according to the Illumina’s sample preparationprotocol and too much different kit used and ı dont find what is wrong with. '' A new approach to determining whole viral genomic sequencesincluding termini using a single deep sequencing run''
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