title: GSM4419660: Tratamiento B2; Mus musculus; miRNA-Seq
library_construction_protocol: We lysed B16F1 cells with TRIzol reactive (Invitrogen) and collected the lysate in 1.5 mL tubes. To solubilize the melanin, we heated for 2 min at 65°C. By successive centrifugations with chloroform, isopropanol, and 75% ethanol, the pellet was obtained and dried at room temperature for 10 minutes. The extracted RNA was re-suspended in transport buffer following the manufacturer's recommendations. For RT-qPCR, total RNA was re-suspended in H2O DEPC and quantified by spectrophotometry. To obtain enriched fractions of small RNAs (≤ 200 nucleotides) was used miRVanaTM miRNA Isolation Kit (Ambion, Austin, USA) following the manufacturer's recommendations. We evaluated the quality of RNA by RIN number (RNA integrity number). To build the library, it was through TruSeq Small RNA Library Prep. Kit (Illumina) following the manufacturer's recommendations. For this, a fraction of total RNA was coupled to 5` and 3` adapters to perform the corresponding RT-PCR for 15 cycles. The products of the amplification were separated by 6% polyacrylamide gel electrophoresis, and the region of the amplified library containing the fraction of miRNAs (from 140-150 nucleotides), was separated, precipitated with ethanol and quantified on nanodrop. Selected libraries were pooled and run on an Illumina Hiseq 2500, single read (SR 1*50) format to generate 1.0 * 106 readings per sample.
attributes.tag: GEO Accession
attributes.value: GSM4419660