GSM3559255: DT40-ASF_Untransfected; Gallus gallus; RNA-Seq
Sureselect strand specific mRNA library preparation for illumina platform sequencing. Libraries for RNA sequencing were prepared by Illumina compatible SureSelect strand-specific RNA Library prep kit (Agilent) at the Genotypic Technology Pvt. Ltd., Bangalore, India. mRNA enrichment was performed from 1 µg of Qubit quantified total RNA. The enriched mRNA was fragmented for 4 min at 94°C in the presence of divalent cations. Synthesis of the first strand cDNA was performed in the presence of actinomycin-D (Gibco) and purified using HighPrep magnetic beads (Magbio Genomics Inc.). Purified ss cDNA was used for second strand synthesis followed by the end repair using second strand synthesis containing end repair mix. The end-repaired cDNA was purified by using HighPrep magnetic beads. Adapters were ligated to the cDNA molecules after 3' adenylation. The adaptor-ligated DNA was purified with HighPrep beads and then amplified for 10 cycles of PCR using Illumina-compatible indexing primers provided in the SureSelect strand-specific RNA library prep kit. The final PCR product was purified with HighPrep beads, followed by library quality control check. The sequencing library was initially quantified by Qubit fluorometer (Thermo Fisher Scientific) and its fragment size distribution was analyzed on the Agilent TapeStation system. Finally, the sequencing library was quantified accurately by quantitative PCR using the Kapa Library Quantification Kit (Kapa Biosystems). The PCR quantified libraries were then pooled in equimolar amounts to create a final multiplexed library pool for sequencing on an Illumina sequencer for 75 base paired-end chemistry.
GEO Accession
GSM3559255