title: GSM4703720: H6 K; Mus musculus; RNA-Seq
library_construction_protocol: Illumina: Total RNA samples were prepared for sequencing using the Qiagen Allprep total RNA kit (Qiagen). Briefly, tissue sections (20-30µg) taken from the middle of the kidneys were lyzed and homogenized in 600 µl Buffer RLT in 2 mL Magna Lyser green beads tubes (Roche Life sciences, Norway) at 600 rpm for 30 sec using the Precellys 24 tissue homogenizer (Bertin Thechnologies, France). The lysates were centrifuged at full speed for 3 min, the supernatant was carefully transferred to the AllPrep spin column, and RNA isolation was performed according to the manufacturer's protocol. The Isolated RNA were analyzed by Agilent. Only RNA samples of high quality (RNA Integrity number (RIN) ≥8.0) were used. Ion Torrent: Total RNA of lymph nodes of four and eight weeks, proteinuric mice, immune aggregates, dissected manually under the loupe (magnification:1:10) of protein uric kidney and kidney tissue from the same kidney purified by TRIzol regent (Ambion, Thermo Fisher, Oslo, Norway) according to manufacturer's instructions. Total RNA of lymph nodes from anti-dsDNA antibody positive mice were isolated using miRNeasy mini kit (Qiagen, Oslo, Norway). The concentration and quality of extracted total RNA were assessed using the AgilentR RNA 6000 nano kit with the AgilentR 2100 BioanalyzerR instrument (Agilent, Matriks AS, Norway). All RNA used in this study had RNA integrity value (RIN value) ≥ 7. Illumina: RNA libraries were prepared for sequencing using standard Illumina protocols Ion Torrent: Polyadenylated (poly(A)) mRNA was isolated from total RNA using Dynabeads mRNA Direct Micro Kit (Ambion, Thermo Fisher) followed by its fragmentation using RNase III with an average sizes of 100-200 nt. The fragmented mRNA was cleaned up and determined the yield and size distribution using the AgilentR RNA 6000 Pico kit with the AgilentR 2100 BioanalyzerR instrument. After adapter ligation at both ends of fragmented mRNA, performed reverse transcription, followed by PCR amplification.  After purification of amplified cDNA analyzed to assess the yield and size distribution using AgilentR High Sensitivity DNS kit with AgilentR 2100 BioanalyzerR instrument.
attributes.tag: GEO Accession
attributes.value: GSM4703720