Assay summary for RNA-seq_Yang_Lab_0001AS

Assay Type:
Yang Lab, Shanghai Chenshan Botanical Garden
A protocol/kit available from Life Technologies with minor modifications was used to construct mRNA-seq libraries. First, the total RNA was estimated on an Agilent 2100 Bioanalyzer (Agilent Technologies,Waldbronn,Germany). Second, The ribosomal RNA-removal steps were replaced by two rounds of polyA purification, with the first round using the PolyATtract® mRNA Isolation Systems (Promega, Madison, WI, USA) and the second round using the Poly(A)Purist™ Kit (Ambion, Austin, TX, USA). About 0.8 μg of mRNA was fragmented with 10min at 37 oC RNase III treatment. The fragmented mRNA were ligated with adaptor Mix A, subsequently used for reverse transcription. The first strand cDNA were separated using 6% TBE-Urea Gel (Invitrogen, Carlsbad, CA, USA) and 100–200 nt fraction was recovered. The fractionated cDNA were subjected to 11-15 cycles of PCR amplification, with the PCR products purified to yield the SOLiD Fragment Library ready for emulsion PCR. Emulsion PCR was performed using 600 pg of the library. All experiments were performed on full sequencing slides.
Library Preparation:
Added by User:
Date Added:
May 9, 2017, 2:38 a.m.
Last Edited:
Oct. 19, 2018, 8:32 a.m.