GSM6591749: heart_E10.5_WT; Mus musculus; RNA-Seq
The tissues obtained from emryonic day 10.5 heart were treated with cleanroom wipers. After removing blood stains on the tissue surface, resh tissue was embedded with OCT then snap frozen at -80℃. Cryosections were cut at 10-µm thickness, mounted onto the ST arrays .Next, the tissue was dehydrated with isopropanol for 1 min followed by staining with H&E. Slides were mounted in 80% glycerol and brightfield images were taken on a Leica DN6B at 20× resolution Library preparation slides used were purchased from the Spatial Transcriptomics team (https://www.10xgenomics.com/), Each of the spots printed onto the array is 55µm in diameter and 100µm from center to center, covering an area of 6.5×6.5 mm2.Each slide Includes 4 Capture Areas, each with ~5,000 unique gene expression spots.After brightfield imaging, the ST slide was prewarmed to 42 °C and attached to a microarray slide module (Grace Biolabs). i. Add 70 μl Permeabilization Enzyme to B1 along the side of the well (30 min) and tap cassette.ii. Apply Slide Seal to the Slide Cassette and place on the Thermocycler Adaptor at 37°C. Close lid. iii.After 6 min, remove the Slide Cassette from Thermocycler Adaptor and remove seal. iv.Add 70 µl Permeabilization Enzyme along the side of the well to the C1 and incubate on the Thermocycler Adaptor at 37°C with the lid closed. v. Repeat process, working backwards to the shortest incubation time (3 min). Remove Slide Cassette from the Thermocycler Adaptor after time course completion.Using a pipette, remove Permeabilization Enzyme from each well without touching the tissue sections.Add 100 µl 0.1X SSC to all wells except the positive control. Using a pipette, remove 0.1X SSC from each well. Add 50 µl Fluorescent RT Master Mix to each well, ensuring that the well surface is uniformly covered with the Master Mix.Apply Slide Seal on the Slide Cassette firmly and place on the Thermocycler Adaptor on the pre-heated thermal cycler. Close thermal cycler lid(53°C 45min) then Skip Pre-equilibrate to initiate cDNA Synthesis. Libraries were prepared after the above operations. Libraries were diluted to 4 nM and paired-end sequencing was performed on an Illumina NovaSeq 6000 sequencer, with 31 cycles for read 1 and 55 cycles for read 2. Between 100 million and 200 million raw read-pairs were generated for each sequenced library.