GSM6647927: Mouse_E13.5_Asxl3+/+_3; Mus musculus; RNA-Seq
RNA was prepared following the Seq-Well protocol (Aicher et al., 2019, Hughes et al., 2020). Briefly, functionalized Seq-Well arrays were loaded with barcoded beads. 20,000 cells were loaded onto the arrays and incubated for 15 minutes then washed with PBS. Functionalized membranes were applied on top then sealed in an Agilent clamp, before incubating at 37℃ for 45 minutes. Sealed arrays were incubated in a lysis buffer  for 20 minutes followed by a 45 minute incubation with hybridization buffer. Beads were removed from arrays by centrifuging at 2000xg for 5 minutes in wash buffer. To perform reverse transcription beads were incubated with the Maxima Reverse Transcriptase for 30 minutes at room temperature followed by overnight incubation at 52℃. Reactions were treated with Exonuclease 1  for 45 minutes at 37℃. Whole transcriptome amplification was performed using the 2X KAPA Hifi Hotstart Readymix . Beads were split into 1,500-2,000 beads per reaction and run under the following conditions 4 Cycles (98℃:20s, 65℃:45s, 72℃:3m) 12 Cycles (98℃:20s, 67℃:20s, 72℃:3m) final extension (72℃:3m, 4℃:hold). Products were purified with Ampure SPRI beads at a 0.6X volumetric ratio then a 1.0X volumetric ratio. Libraries were prepared following the Seq-well protocol (Aicher et al., 2019, Hughes et al., 2020) and using the Nextera XT kit.