title: GSM8122769: LCM_RNA_CTX; Mus musculus; OTHER
library_construction_protocol: For embryo collection, pregnant dams were euthanised by CO2 overdose with exposure to gas in rising concentration followed by cervical dislocation. Embryos were euthanised by cooling on ice followed by decapitation. Heads were frozen in OCT resin by immersion in a dry ice/isopentane bath followed by storage at -80C. Cryosections were cut using a XXXX cryostat under RNAse free conditions. The instrument was wiped with RNAse-ZAP prior to use and between different blocks. A different blade was used for each block. Sample blocks were equilibrated to the chamber temperature for 15 minutes prior to sectioning, and then mounted on the object holder. Sections were cut at a thickness of 10 uM and collected on RNAse-free SuperFrost Plus microscope slides (one section per slide). Slides were left at room temperature for 20-30 seconds to help tissue adhesion and then stored on dry ice (short term) or at -80C (long term). Tissue sections from e16.5 mouse embryos (as described in the “Animal housing” section) were cut as described in the “Tissue collection” section. Sections were stained with Haematoxylin/Eosin using the following fast procedure: 1) 40 seconds in 75% ethanol, 2) 30 seconds in mQ water, 3) 30 seconds in Harris' modified Haematoxylin solution (Sigma HHS16), 4) 3x 30 seconds in mQ water, 5) 30 seconds in a 1:1000 dilution o 28% Ammonium Hydroxide (Sigma 338818) in mQ water, 6) 10 seconds in 5% Eosin Y solution (Sigma 318906), 7) 30 seconds each in 70%, 95% and 100% ethanol, 7) 2x 20 seconds in Xylene. The stained sections were imaged and cut on a Leica LMD6000 LCM microscope. Areas corresponding to the sub-ventricular zone and to the cortex (matching the illumination areas for the BALI experiment) were used for cutting. Tissue fragments were collected in the caps of 0.5ml RNAse-free tubes in 50ul of lysis solution from the RNAqueous micro RNA extraction kit (Thermo Scientific). RNA extraction was performed according to the RNAqeous micro kit protocol for LCM extraction. In short: 1) 50ul of lysis buffer were added to the 50ul including the sample, and the tube was incubated at 42C for 30 minutes, 2) the collection column was pre-wetted with 30ul lysis solution, 3) 3ul LCM additive were added to each sample, 4) 129ul of 100ul Ethanol were added to each sample, 5) the samples were bound to the collection column, and the column was washed with 180ul wash solution 1 and twice with 180ul of wash solution 2/3, 6) RNA was eluted with a 5 minute incubation step with 10ul of pre-heated (95C) elution solution. Purified RNA was treated with DNAse1 using the optional DNAse treatment step in the RNAqueous kit: 1) 1ul of DNAse buffer and 1ul of DNAse I enzyme were added to each sample, 2) samples were incubated for 20 minutes at 37C, 3) 2ul of DNAse inactivation reagent were added to each sample, and the inactivated DNAse was removed by centrifugation. RNA abundance and quality were evaluated by Agilent Tapestation (DNA5000 High-Sensitivity screentape). RNA sequencing libraries were prepared using the NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina (NEB) according to the recommended protocol using 15ng RNA as input material. Library quality and abundance was measured by Tapestation (DNA5000 screentape) and qPCR (KAPA Illumina library quantification kit). Libraries were sequenced on a NextSeq500 instrument using paired end 75nt reads.