title: GSM4996433: Human-Cerebellum-Mock Rep2; Homo sapiens; RNA-Seq
library_construction_protocol: RNA purchase/extraction:  Human Adult Normal Brain Total RNA (R1234035-50), Human Adult Normal Cerebellum Total RNA (R1234039-50), Rat Brain Total RNA (R1434035-50), Monkey(Rh) Brain (R1534035-50) Total RNA was obtained by BioChain. Human Blood Total RNA (HR-705) was obtained from Zyagen.One C57BL/6 male and one female were sacrificed at p21, different brain regions were harvested (cerebellum, cortex, olfactory bulb, brain stem and, thalamus) by the Nelson's lab at Brandeis University. The RNA was extracted with TRI reagent (Sigma, Aldrich) , followed by Zymo RNA cleaning and concentrator -25 Kit (Zymo), according to the manufactures' instruction. We collected and dissected 10 days old D. melanogaster (Canton-S) heads and brains. Total RNA was  extracted with TRI reagent (Sigma, Aldrich), followed by DNase (DnaseI, NEB) treatment. Recovered RNA was used to prepare a total RNA library, adapting the protocol from Shishkin et al. , Nature 2015. Specifically, we prepared one library for the RNAseR treated samples and one for the Mock ones. Briefly, RNA was fragmented for 3 min at 92°C in FAstAP 2X buffer (ThermoFisher). Subsequently, samples were dephosphorylated with FastAP enzyme (ThermoFisher), the first linker was ligated, and samples belonging to the same library were pulled together. Retro-transcription was performed using Affinity Script (Agilent). After second ligation, we performed PCR to add the external barcode. For Human Blood RNA samples and D. melanogaster samples, libraries were prepared using the NEXTFLEX® Rapid Directional RNA-Seq Kit 2.0 by PerkinElmer, following the manufacturer's instructions. RnaseR vs Mock ribodepleted total RNA library The samples were sequenced by Novogene Corporation Inc.(8801 Folsom BLVD, Suite 290, Sacramento, CA 95826)- HiSeq 1% PhiX with HiSeq-4000sequencer