title: GSM5287946: 26_612_FGF13_sh13; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted from cell cultures using an RNeasy Mini Kit (Qiagen, Germany) according to the protocol and DNAse treated in the RNEasy column. The quality of the total RNA samples was ensured with Agilent Bioanalyzer 2100 and Advanced Analytical Fragment Analyzer. Sample concentration was measured with Nanodrop ND-2000, Thermo Scientific. The library preparation was started from 100 ng of total RNA. Library preparation was done according to Illumina TruSeq® Stranded mRNA Sample Preparation Guide (part # 15031047): The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligo attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity is achieved by replacing dTTP with dUTP in the Second Strand Marking Mix (SMM), followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. The incorporation of dUTP in second strand synthesis quenches the second strand during amplification, because the polymerase used in the assay is not incorporated past this nucleotide. The addition of Actinomycin D to First Stand Synthesis Act D mix (FSA) prevents spurious DNA-dependent synthesis, while allowing RNA-dependent synthesis, improving strand specificity. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter. The products are then purified and enriched with PCR to create the final cDNA library. Unique Illumina TruSeq indexing adapter was ligated to each sample during adapter ligation step to be able to later pool several samples in one flow cell lane.