GSM8646189: 10, skin, allergic contact dermatitis, methylisothiazolinone; Homo sapiens; RNA-Seq
Total RNA was extracted from  skin biopsies with Genelute mammalian total RNA kit from Sigma- Aldrich (cat. No. RTN350). Tissue was disrupted and digested in the Lysis buffer with proteinase K (P4850, Sigma-Aldrich) and 2-mercaptoethanol (M3148, Sigma-Aldrich) incubating it for 10 minutes at 55ºC.  After washing and centrifugation several times to remove contaminants, RNA was eluted with 50 μl of Elution buffer. Two commercial kits were tested. This kit obtained the best performance in total RNA recovered and integrity of the sample. The samples were used immediately or stored at -80ºC for future use. Samples were processed according to Truseq RNA Exome protocol from Illumina (Document #10000000039582 v01 March 2018) following steps from 1 to 6_Enrich DNA Fragments page 18. Afterwards, samples were processed using Illumina RNA preparation with enrichment Tagmentation protocol (Document #1000000124435 v03 April 2021) following steps from 6_Normalize Library page 15 to the end of the protocol. Samples were enriched with Illumina Exome Panel and Unique Dual Index from NEBNext were used. The initial total RNA input for every sample was 100 nanograms measured with Qubit 4.0. 200 nanograms of each amplified sample was enriched in pools up to 3 samples, libraries profiles were checked by 2100 Bioanalyzer. All samples amplified enough to continue with the process. Final enriched libraries were quantified by 2100 Bioanalyzer (DNA 1000 kit). Samples were sequenced in a NextSeq2000 from Illumina with an estimated output of 25 million reads/sample and a read length of 2x50bp. Exome RNA capture