GSM4592593: CS17_DL_1; Homo sapiens; RNA-Seq
Human embryos embedded in OCT were sectioned in a caudal-to-rostral direction using a cryotome FSE cryostat (Thermo Scientific).  Sections were stained using a rapid Haematoxylin and Eosin staining protocol - 3 minutes (min) in 70% ethanol, 1 min H2O, 4 min Mayer's Haematoxylin Solution (Sigma-Aldrich), 2 min tap H2O, 15 seconds Eosin Y (Sigma-Aldrich), 1 min 70% ethanol, 1 min 90% ethanol, 3 mins 100% ethanol. All H2O was treated with diethyl pyrocarbonate (DEPC) and all reagents were pre-cooled in ice except 100% ethanol which was room temperature. The laser capture microscope used was the PALM microbeam (Zeiss). The microscope and surrounding area were sprayed down with RNaseAWAY ® (Sigma-Aldrich). Sections were viewed and microdissected in brightfield using a 10X objective. The microdissected regions were collected into the caps of AdhesiveCap 500 opaque (Zeiss) 500µl PCR tubes. 15µl lysis buffer (0.2% Triton X-100 (Sigma-Aldrich), 2U/µl RNase inhibitor (Takara), Phosphate Buffer Solution (PBS, (Nichterwitz et al., 2016)) was added directly on top of the dissected tissue in the tube caps and the tubes closes in an inverted position. PicoPure RNA Isolation Kit was used to extract RNA for samples 1-12. Samples 13-30 were directly amplified from lysed material SMARTer® Stranded Total RNA-Seq Kit v2 -Pico Input Mammalian kit (Takara) for samples 1-12, LCM-Seq (Picelli et al., 2014) for samples 13-30.
GEO
GSM4592593