GSM2053609: 2776-TL-0052 miRNA; Mus musculus; miRNA-Seq
After 48 hr of seeding on substrates, cells were lysed using QIAzol Lysis Reagent (Qiagen Sciences, Germantown, MD USA) following the manufacturer’s protocol. The cell lysate was immediately stored at -80°C and transported on dry ice to the Genomic Services Laboratory (HudsonAlpha, Huntsville, AL, USA) for RNA isolation and sequencing. Total RNA containing both mRNA as well as microRNA fractions was extracted from the cell lysate using the miRNeasy Kit with on-column DNase treatment (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's protocol. The final elution was performed in 30 µl of RNase-free sterile distilled water. The concentration and integrity of the extracted total RNA were estimated using the Qubit® 2.0 Fluorometer (Invitrogen)and Agilent 2100 Bioanalyzer (Applied Biosystems, Carlsbad, CA, USA), respectively. RNA samples with RNA integrity numbers (RIN) of at least 7.0 were used for further processing. Total RNA from each sample was taken into small RNA library preparation protocol using NEBNext Small RNA Library Prep Set for Illumina (New England BioLabs Inc., Ipswich, MA, USA) according to manufacturer’s protocol. Briefly, 3` adapters were ligated to total input RNA followed by hybridization of multiplex SR RT primers and ligation of multiplex 5` SR adapters. Reverse transcription (RT) was done using ProtoScript II RT for 1 hour at 50°C. Immediately after RT reaction, PCR amplification was performed for 15 cycles using LongAmp Taq 2X master mix. Illumina indexed primers were added to uniquely barcode each sample. Post- PCR material was purified using QIAquick PCR purification kit (Qiagen Inc., Valencia, CA, USA). Post-PCR yield and concentration of the prepared libraries were assessed using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, California, USA) and DNA 1000 chip on Agilent 2100 Bioanalyzer (Applied Biosystems, Carlsbad, CA, USA), respectively. Size selection of small RNA was done using a 3% dye free agarose gel cassettes on Pippin prep instrument (Sage Science Inc., Beverly, MA, USA). Post-size selection yield and concentration of the libraries were assessed using Qubit 2.0 Fluorometer and DNA High sensitivity chip on Agilent 2100 Bioanalyzer, respectively. Accurate quantification for sequencing applications was performed using the qPCR-based KAPA Biosystems Library Quantification kit (Kapa Biosystems, Inc., Woburn, MA, USA). Each library was diluted to a final concentration of 1.25nM and pooled in equimolar ratios prior to clustering. Single End (SE) sequencing (50bp) was performed to generate approximately 15 million reads per sample on an Illumina HiSeq2500 sequencer (Illumina, Inc., San Diego, CA, USA).
GEO Accession
GSM2053609