GSM4161121: EAE Th1* replicate 2; Mus musculus; RNA-Seq
200ul of chloroform/1000ml total volume, incubate 3 min. RNA quality was assessed using Pico Bioanalyser chips (Agilent). RNASeq libraries were prepared using the Clontech Smart-Seq Ultra low RNA kit (Takara Bio) for cDNA preparation, starting with 550 pg of total RNA, and 13 cycles of PCR for cDNA amplification. The SMARTer Thruplex DNA-Seq kit (Takara Bio) was used for library prep, with 7 cycles of PCR amplification, following the manufacturer's protocol. The amplified library was purified using AMPure beads, quantified by Qubit and QPCR, and visualized in an Agilent Bioanalyzer. The libraries were pooled equimolarly, and run on a HiSeq 4000, as paired end reads, 50 nucleotides in length. Precipitate with one volume of 100% isopropanol. Leave @ -20 for > 1 hr.  Spin down @ full speed, 30min, 4oC. Gently aspirate with a pipet tip and resuspend in freshly prepared 75% ETOH. Mix by inverting the tube.  Spin down @ full speed, 5min, 4oC. Gently aspirate with a pipet tip and resuspend thoroughly in up to 87.5ul of prewarmed H2O. Add 10ul of buffer RDD and 2.5ul of resuspended DNAse solution. Incubate for 10minutes @ RTemp.  Add b-ME to buffer RLT à [3.5ul per 350ul of RLT]. Then, add 350ul of buffer RLT and mix very well.  Add 250ul of 100% ETOH and mix very well by pipetting.  Add 700ul to RNeasy minElute column. Pipette out the flow through rather than decanting to avoid salt accumulation around the sides of the collection tube. Reuse the collection tube for the next step. Pipet 500 μl Buffer RPE onto the RNeasy MinElute spin column. Close the lid gently and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the column. Discard the flow-through and reuse the collection tube in the next step. Pipet 500 μl of freshly prepared 80% ethanol onto the RNeasy MinElute spin column. Close the lid gently and centrifuge for 2 min at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the collection tube with the flow-through. After centrifugation, carefully remove the RNeasy MinElute spin column from the collection tube to avoid the carryover of ethanol.   Place the RNeasy MinElute spin column into a new 2 ml collection tube (supplied). Open the lid of the spin column, and centrifuge at full speed for 5 min to dry the membrane. Discard the collection tube with the flow-through. Place the RNeasy MinElute spin column in a new 1.5 ml collection tube (supplied). Add 14 μl RNase-free water directly to the center of the spin column membrane. Close the lid gently, and centrifuge for 1 min at full speed to elute the RNA.
GEO Accession
GSM4161121