GSM2902386: day7_DM2_rep2; Mus musculus; RNA-Seq
RNA was isolated with ExtractRNA reagent (#BC032, Evrogen, Moscow, Russia), quantified with Bioanalyser 2100 instrument (Agilent Technologies, Palo Alto, CA) using BA RNA nano kit; concentration was determined on Nanodrop 1000 (Thermo Scientific, Wilmington, DE, USA). Full-length poly(A) mRNA was isolated with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB E7490). RNA fragmentation, first and second strand synthesis, end repair, adaptors ligation, and PCR enrichment with Indexed primers (NEBNext® Multiplex Oligos for Illumina, NEB E7335S) were performed using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB E7420), according to manufacturer manual. For first strand synthesis, Actinomycin D (Sigma A1410) was used to inhibit DNA-dependent DNA synthesis. After enrichment, PCR amplification with 11 cycles on Veriti Thermal Cycler was performed (Applied Biosystems, Foster City, CA, USA), followed by purification with Agencourt AMPure XP Beads (Beckman Coulter, Inc.). Libraries were quantified with Bioanalyser 2100 (Agilent Technologies, Palo Alto, CA) instrument using BA High sensitivity kit, accurate molarity quantification was obtained with qPCR according to Illumina Sequencing Library qPCR Quantification Guide using standard qPCR primers 1.1 and 1.2, and qPCRmix-HS SYBR+LowROX kit (#PK156L, Evrogen , Moscow, Russia), using 7500 real-time PCR system (Applied Biosystems, Foster City, CA).
GEO Accession
GSM2902386