Transcriptome Sequencing

Sample Requirement:

  • Snap Frozen Tissue Samples to be provided in dry ice.

Or,

  • RNA sample  ≥2μg  [RIN Value – ≥6.8(animals) / 6.3 (plants & fungi) ] ;   Volume-  20 ul,  Concentration – ≥50ng/ μl,

 

Sample Quality Control:

  • NGS grade High quality RNA will be isolated from the supplied sample using commercially available kits.
  • The qualification and quantification of RNA will be carried out by denatured agarose gel electrophoresis and NanoDrop/Qubit Fluorometer analysis.

 

Library Preparation:

  • RNA Seq library will be prepared by using TruSeq Stranded mRNA Sample Prep Kit or TruSeq Stranded Total RNA Library Prep Kit.
  • Preparation of indexed RNA library by enriching mRNA from Total RNA for sequencing on Illumina Platform.
  • Library validation will be carried out using High Sensitivity D1000 Screen tape using Agilent Tape Station.

 

Sequencing:

  • Sequencing will be carried out on Illumina Novaseq platforms with 2×150 for RNA v2 chemistry.
  • Generation of data as per requirement.

 

Bioinformatics Analysis:

  • Standard Analysis (Agricultural part-with reference）
  • Data Quality Control: filtering reads containing adapter or with low quality
  • Statistics Analysis of Data Production and Quality
  • Mapping Reads to Reference Genome
  • Novel Gene Prediction
  • Alternative Splicing Analysis
  • SNP/In Del Analysis
  • Identification of anti-Transcript (Strand Specific Library Only)
  • TSS and TSS Prediction (Strand Specific Library Only)
  • Gene Expression Quantification
  • Correlation analysis (For biological replicates only)
  • Differential Expression Analysis (two or more groups of samples)
  • GO Enrichment Analysis and KEGG Pathway Enrichment Analysis of Differentially Expressed Genes (DEGs) (two or  more groups of samples) 
  • Protein to Protein Interaction Analysis
  • Transcription Factor Analysis of Differentially Expressed Genes (DEGs) (two or more groups of samples) (for plants only)
  • Transcription Factor Analysis (for animals only)

 

Delivery Time :

  • Provided that there are no unexpected difficulties (biological or technical), 8-9 weeks from the date of QC qualified samples received. All samples to be provided in a single lot.