title: GSM8821216: iTF_Microglia_RPF_Nontargeting_Rep2; Homo sapiens; RNA-Seq
library_construction_protocol: For RNA samples, cells were lysed with Trizol and RNA was extracted using phenol/chloroform. For RPF samples, cells were harvested in cyclohexamide and lysed and treated with RNase I. Lysates were subjected to a sucrose gradient and the polysome pellet was resuspended in Trizol. RNA extracted using phenol/chloroforml. For RNA samples, libraries were constructed using the Illumina Stranded mRNA Ligation preparation kit. In brief, equal amounts of RNA are subjected to polyA selection and subsequent fragmentation. Samples are reverse transcribed and adapters are ligated. The products are then purified and amplified for sequencing. For RPF samples, linkers are ligated to ribosome protected RNA fragments and subsequently reverse transcribed and gel purified. After purification, products are circularized and subjected to rRNA depletion prior to amplification with sequencing barcodes.