title: GSM4365362: DP-3_6; Mus musculus; RNA-Seq
library_construction_protocol: Brains were removed and frozen in embedding matrix before dissection of dorsal raphe (DR) from 4 consecutive 300 um-thick coronal sections (Bregma -4.0 mm to Bregma -5.2 mm, area ~ 1.5x1.5 mm2 directly below cerebral aqueduct). Tissue was stored in RNAlater (ThermoFisher Scientific) until further processing. For RNA extraction, Qiagen's miRNEasy Mini kit was used: Tissue was placed into 700 µl Qiazol (Qiagen) and mechanically disrupted until the homogenate was clear. This mix was incubated at room temperature (RT) for 5 min before addition of chloroform, with a further incubaton period of 3 min after agitating the solution vigorously. After centrifugation (15 min, 12000 G, 4 °C), the upper phase was transferred and RNA was precipitated with 1.5 × the sample volume in ethanol (96 %). Provided columns and collection tubes were used to retain the precipitate prior to washing the filter with the provided wash buffers (RWT once, RPE twice). At each of these steps, the flowthrough after a quick centrifugation step was discarded. Finally, RNA was eluted from the column filter using 40 µl of RNAse-free water. NEBNext® Ultra™ II RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA)