Publication protocol
Enriched small-RNA fractions isolated from infected and uninfected HEK293 cells were used to construct libraries of mature miRNAs and pre-miRNAs. A mature miRNA library was generated using a Truseq small-RNA preparation kit (Illumina, USA) according to Illumina's sample preparation guide. The purified cDNA library was used for cluster generation on Illumina's Cluster Station and then sequenced on an Illumina GAIIx following the vendor's instructions. Raw sequencing reads (40 nt) were obtained using Illumina's Sequencing Control Studio software version 2.8 (SCS v2.8) following real-time sequencing image analysis and base calling with Illumina's Real-Time Analysis version 1.8.70 (RTA v1.8.70). A proprietary pipeline script, ACGT101-miR v4.2 (LC Sciences, USA), was used to trim the adaptor sequences, remove various unmappable sequencing reads, and map the remaining unique reads with the reference database files of mature miRNAs (ftp://mirbase.org/pub/mirbase/CURRENT/). The RNA library for pre-miRNA sequencing was prepared using an mRNA-Seq Sample Prep kit (Illumina, USA), and sequencing was conducted on an Illumina HiSeq 2000 instrument according to the manufacturer's protocol. The 3′ adaptor for sequencing was removed from the raw reads, and reads with a length of less than 10 nt were excluded. The remaining reads were aligned with human pre-miRNAs (ftp://mirbase.org/pub/mirbase/CURRENT/) using Bowtie v1.0.0. The number of read copies from each sample was tracked during mapping of mature miRNAs and pre-miRNAs and normalized for comparison. The ACGT101-miR v4.2 script (LC Sciences, USA) was then used to perform the differential-expression analysis. Normalization of sequence counts in each sample (or data set) was achieved by dividing the read counts by a library size parameter of the corresponding sample. The library size parameter was a median value of the ratio between the counts in a specific sample and a pseudo-reference sample. A count number in the pseudo-reference sample was the geometric mean count across all samples. Library preparations, sequencing, and bioinformatic analysis were performed by LC Sciences, LLC (Houston, TX, USA).
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