title: A Human Fetal Cochlear Cell Atlas Reveals a Regulatory Blueprint for Spatial Patterning- Stereo-seq
design: A Human Fetal Cochlear Cell Atlas Reveals a Regulatory Blueprint for Spatial Patterning- Stereo-seq
library_construction_protocol: Fresh human fetal cochlear samples at 10, 14, and 16 post-conceptual weeks (PCW) were collected.  Tissues were rinsed with PBS, blotted dry, embedded in OCT compound, and flash-frozen in liquid nitrogen. Using a Leica CM1950 cryostat, the embedded samples were sectioned at a thickness of 10 μm.  Sections with intact cochlear morphology and optimal architecture were selected under microscopic guidance for subsequent processing. Tissue sections on chips were permeabilized in 0.01 M HCl supplemented with 0.1% pepsin at 37°C for 12 minutes to release RNA. The released RNA was reverse-transcribed using a Stereo-seq-specific TSO oligonucleotide to generate first-strand cDNA. After tissue removal and Exonuclease I treatment, cDNA was amplified (15 cycles). The amplified product was fragmented using Tn5 transposase and then further amplified (13 cycles) with Stereo-seq library primers to construct the final sequencing library. Library purification was performed using VAHTS™ DNA Clean Beads.