GSM1315486: Microglia-RNAseq-wt; Mus musculus; RNA-Seq
RNA was purified using RNeasy Mini Kit (Qiagen) and enriched for Poly(A)-RNA with MicroPoly(A) Purist Kit (Ambion). Subsequently, RNA was treated with TURBO DNase (Ambion), fragmented with RNA Fragmentation Reagents (Ambion) and purified by a P-30 column (Biorad). Fragmented RNA was dephosphorylated with Antarctic phosphatase (New England Biolabs) heat inactivated and precipitated over-night. Poly(A)-tailing and cDNA synthesis was performed as previously described. For reverse transcription, oligos with custom barcodes (underlined) were used: 5′-Phos-CA/TG/AC/GT GATCGTCGGACTGTAGAACTCT/idSp/CAAGCAGAAGACGGCATACGATTTT TTTTTTTTTTTTTTTTVN-3′. Subsequently, exonuclease was used to remove the excess oligo. After heat-inactivation, RNA was hydrolyzed by alkaline treatment (100mM NaOH) and heat at 95°C for 25 min. The cDNA fragments of 50–150 nucleotides were purified on a denaturing Novex 10% polyacrylamide TBE-urea gel (Invitrogen). The recovered cDNA was circularized, linearized, amplified for 12 cycles, and gel purified as previously described. The library was sequenced on Illumina sequencers according to the manufacturer's instructions.