GSM4903875: H16 V1 N; Homo sapiens; RNA-Seq
The pellet was dissolved in 1 ml of TRIzol™ and stored at -80°C until RNA was isolated. After all samples were obtained, they were thawed at room temperature and, after 200 µl of chloroform was added, shaken vigorously for 30 seconds. The samples were kept at RT for 10 mins and centrifuged at 16,000g for 15 mins at 4°C. The aqueous phase was cleaned up and concentrated with protocol 5.3 using the Nucleospin® RNA XS extraction kit (Macherey-Nagel). No carrier RNA was added as it would interfere with RNA sequencing. The NEBNext Ultra Directional RNA library prep kit for Illumina was used to process the samples. The samples preparation was performed according to the protocol “NEBNext Ultra Directional RNA Library Prep Kit for Illumina” (NEB #7420S/L). Briefly, oligo-dT magnetic beads were used to isolate mRNA from total RNA. cDNA synthesis was performed after fragmentation of this mRNA. This was used for ligation with sequencing adapters followed by PCR amplification of the resulting product. The quality and yield after sample preparation was measured with the fragment analyser. The size of the resulting products was consistent with the expected size distribution between 300-500 base pairs. To evaluate the quality of the library preparation and kits used, the raw data was sampled and mapped to annotated genomic references. Mapping positions were classified as intragenic, exonic, intergenic, intronic and rRNA. Clustering and DNA sequencing was performed according to the manufacture's protocol using the Illumina NextSeq 500. A concentration of 1.6pM was used as input. The reads were trimmed for adapter sequences using Trimmomatic v0.30 before the alignment. Presumed adapter sequences were removed from the read when the bases matched a sequence in the adapter sequence set (TruSeq adapters) with 2 or less mismatches and an alignment score of at least 12.