GSM8676454: Stereo-CITE-seq, mouse thymus, Protein; Mus musculus; OTHER
Prior to the experiment, all solutions were freshly prepared and maintained on ice. Blocking Buffer (50 µl per sample) was composed of 3x SSC, 0.1% Triton-X-100, 10% serum, 1 µg/µl sheared salmon sperm DNA, 2.5 µl TruStain FcX PLUS (anti-mouse CD16/32) antibody (BioLegend, Cat. No. 156604), 5% RNase inhibitor (RI), and 5 µM 32-nt Poly A. The Wash Buffer consisted of 3x SSC with 5% RI. Cryosectioned OCT-embedded tissue, cut to 10 µm thick, was carefully transferred onto the Stereo-seq chip. These chips, now bearing tissue sections, were immediately warmed to 37℃ for 1 minute using a thermocycler adaptor. Tissues were then fixed by placing the chips in pre-chilled methanol at -20℃ for 30 minutes. Following fixation, blocking was performed by incubating the tissue with Blocking Buffer for 20 minutes, after which the sections were exposed to the TotalSeqTM-A antibody mix in Blocking Buffer for 45 minutes at room temperature. The tissue sections on chips were then washed three times with Wash Buffer, each for 30 seconds. After DAPI staining, the tissue sections were mounted with glycerol and imaged. Subsequent processing followed the Stereo-seq Transcriptomics T Kit protocol with minor adjustments. Briefly, the sections were permeabilized with Permeabilization Mix for an optimized duration, as recommended in the Stereo-seq Permeabilization Kit instructions. After reverse transcription, cDNA and antibody-derived tag (ADT) products were released from the chips using Release Enzyme Mix and collected. Separation of the cDNA fraction from the ADT library was achieved via a 0.8X bead selection, followed by preparation of the cDNA sequencing library in accordance with the Stereo-seq Transcriptomics T Kit. The ADT product in the supernatant was purified using a 2.0X (0.8X + 1.2X) bead selection, then amplified with Protein PCR Primer Mix. Both ADT and cDNA libraries were sequenced using MGI DNBSEQ-T7 sequencing platforms.