GSM3318312: D2_siLINC00941_5; Homo sapiens; RNA-Seq
RNA from the obtained organotypic epidermis was extracted with the QIAGEN RNeasy Plus Mini Kit according to the manufacturer's instructions. Library preparation and mRNA sequencing were carried out according to the Illumina TruSeq Stranded mRNA Sample Preparation Guide, the Illumina HiSeq 1000 System User Guide (Illumina), and the KAPA Library Quantification Kit - Illumina/ABI Prism User Guide (Kapa Biosystems), with minor modifications. In brief, mRNA molecules were purified using oligo-dT probes immobilized on magnetic beads starting with 250 ng of total RNA supplied with ERCC spike-Ins. Chemical fragmentation of the mRNA to an average insert size of 200-400 bases was performed using divalent cations under elevated temperature (94°C, 4 minutes). First strand cDNA was produced by reverse transcription with random primers. Actinomycin D was added to improve strand specificity by preventing spurious DNA-dependent synthesis. Blunt-ended second strand cDNA was synthesized using DNA Polymerase I, RNase H and dUTP nucleotides. The resulting cDNA fragments were adenylated at the 3' ends, the indexing adapters were ligated and subsequently specific cDNA libraries were created by PCR enrichment. The libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit. Equimolar amounts of each library were used for cluster generation on the cBot (TruSeq SR Cluster Kit v3). The sequencing run was performed on a HiSeq 1000 instrument using the indexed, 1x50 cycles single end protocol and TruSeq SBS v3 Reagents according to the Illumina HiSeq 1000 System User Guide. Image analysis and base calling resulted in .bcl files, which were converted into .fastq files by the CASAVA1.8.2 software.