A Human Fetal Cochlear Cell Atlas Reveals a Regulatory Blueprint for Spatial Patterning- Stereo-seq-2
Spatial transcriptomic data was generated from human fetal cochleae of 16 post-conceptual weeks (PCW) using the Stereo-seq platform. Fresh-frozen tissues were sectioned at 10 µm thickness and mounted on pre-chilled capture chips of 0.5×0.5 cm or 1×1 cm sizes. The protocol involved on-chip tissue fixation, permeabilization with 0.1% pepsin, and reverse transcription using a Stereo-seq-specific TSO oligonucleotide. First-strand cDNA was amplified for 15 cycles. Sequencing libraries were constructed via Tn5 transposase fragmentation of 20 ng input cDNA, followed by a 13-cycle amplification with platform-specific primers. Final libraries were sequenced on the MGI DNBSEQ-T7 platform, producing paired-end reads of 35 bp (Read1) and 100 bp (Read2) in length. Raw data was processed through the SAW pipeline (v4.1.0), with reads aligned to the GRCh38 human genome using STAR (v2.5.3). The final output is a spatial gene expression matrix where molecules are quantified by unique Molecular Identifiers (MIs) and mapped to precise spatial locations via Coordinate Identifiers (CIDs).