title: GSM6500732: AR17_MouseBrain_n4_1546ng_WT_E12_LB_pass2; Mus musculus; OTHER
library_construction_protocol: All procedures were conducted in accordance with the Australian National University Animal Experimentation Ethics Committee (protocol number A2019/46). Pregnant females were cervically dislocated, and embryos extracted in cold sterile PBS. The frontal area of the cortex was then dissected with micro-knifes under a Zeiss STEMI 508 stereomicroscope and tissue samples were immediately placed in a 1.5 ml microcentrifuge tube (Eppendorf, DNA) containing 300 µl of denaturing lysis and binding buffer (100 mM Tris-HCl pH 7.4 at 25 °C, 1 % w/v lithium dodecyl sulfate (LDS), 0.8 M lithium chloride, 40 mM EDTA and 8 mM DTT; LBB). Samples were immediately agitated by vigorous pipetting until almost complete tissue dissolution, flash-frozen on dry ice and stored at – 80 °C until sequencing.  150 mg of frontal cortex (PFC) brain region tissue was lysed immediately upon extraction. The tissue/LBB mixture was thoroughly pipetted with 200 µl tip until the sample viscosity was reduced, and pipetting was seamless. 500 µl of oligo(dT)25 magnetic beads (New England BioLabs) suspension was used per replicate. The beads were washed with 1 ml of LBB twice, each time collecting the beads on a magnet and completely removing the supernatant. Upon washing, the oligo(dT)25 beads were resuspended in the tissue/LBB mixture and placed in a rotator set for 20 rpm at 25 °C for 5 minutes, followed by the same rotation at 4 °C for 30 minutes. The suspension was briefly spun down at 12,000 g, separated on a magnet, and the supernatant was discarded. The beads were then resuspended with 1 ml wash buffer (20 mM Tris-HCl pH 7.4, 0.2 % v/v Titron X-100, 0.4 M lithium chloride, 10 mM EDTA and 8 mM DTT; WB) and washed on a rotator set for 20 rpm at 4 °C for 5 minutes, 3 wash rounds in total. The beads were collected on a magnetic rack and the supernatant was discarded. The wash procedure was repeated three times. The elution was carried out stepwise. Washed bead pellet was first resuspended in 50 µl of the elution buffer (25 mM HEPES-KOH, 0.1 mM EDTA; HE). The first suspension was heated at 60 °C for 5 minutes to facilitate the elution, and the eluate was collected upon placing the bead-sample mixture on a magnetic rack, separating the beads, and recovering the clean supernatant. The resultant pellet was next resuspended in another 50 µl of HE buffer, and the process was repeated. The eluates were then combined and subjected to an additional solid-phase reversible immobilization (SPRI) bead purification step and stored frozen. The eluate from oligo(dT) bead extraction was further purified using AMPure XP SPRI beads (Beckman Coulter Life Sciences) according to the manufacturer's recommendations. Briefly, the eluate samples were supplemented with 1.2× volumes of the SPRI bead suspension in its standard (supplied) binding buffer, and the resultant mixture was incubated at room temperature for 5 minutes with periodic mixing. The SPRI beads were brought down by a brief 2,000 g spin down and separated from the solution on a magnetic rack. The supernatant was removed, and the beads were resuspended in 1 ml of 80 % v/v ethanol, 20 % v/v deionized water mixture and further washed by tube flipping. The bead and solution separation procedure were repeated. The ethanol washing process was repeated one more time. Any remaining liquid was brought down by a brief spin and removed using a pipette, and the beads were allowed to air-dry while in the magnetic rack for 2 minutes. The purified RNA was then eluted in 20 µl of deionized water and the RNA content was assessed using absorbance readout via Nanodrop and fluorescence-based detection via Qubit RNA high sensitivity (HS) assay kit (Thermo Fisher Scientific). 