GSM9797113: Sample 2: mouse C57BL/6J, Male, 14 weeks; Mus musculus; RNA-Seq
Total RNA was extracted from a mouse brain tissue sample, with position along the anterior-posterior axis matched to the posterior slices imaged by MERFISH, using TRIzol Reagent (Thermo Fisher) followed by column-based purification with in-column DNase treatment. Briefly, 1 mL of TRIzol was added to the tissue sample and incubated for 5 minutes at room temperature to ensure complete dissociation of nucleoprotein complexes. Phase separation was initiated by adding 0.2 mL of chloroform per 1 mL of TRIzol, followed by vigorous shaking and a 2-3-minute incubation. Samples were centrifuged at 12,000 × g for 15 minutes at 4 oC, and the resulting aqueous phase was carefully transferred to a new tube. An equal volume of 100% ethanol was added to the aqueous phase (1:1), mixed thoroughly, and the solution was applied to a Zymo-Spin™ IC Column (Zymo Research). After centrifugation, the flow-through was discarded. Columns were then washed with 400 µL RNA Wash Buffer. For genomic DNA removal, DNase I treatment was performed in-column by applying 40 µL of freshly prepared DNase I reaction mix (5 µL DNase I enzyme and 35 µL DNA Digestion Buffer) directly onto the column matrix, followed by a 15-minute incubation at room temperature. After DNase treatment, RNA purification continued according to the manufacturer's RNA Clean-up protocol. Columns were washed sequentially with 400 µL RNA Prep Buffer, 700 µL RNA Wash Buffer, and a final wash with 400 µL RNA Wash Buffer. To elute the RNA, 15 µL of nuclease-free water was applied directly to the column matrix and centrifuged. Eluted RNA was stored at -80 oC until further use. Library preparation, quality control, and RNA sequencing were performed by Novogene. Total RNA samples were assessed for integrity and used to construct sequencing libraries following standard Illumina protocols. Libraries were sequenced on an Illumina platform to generate 150 bp paired-end reads. Paired-end FASTQ files were quantified using Salmon against the GENCODE mouse vM25 transcriptome reference. A Salmon index was generated from gencode.vM25.transcripts.fa using k-mer size 31. Transcript-level abundances were estimated using Salmon quasi-mapping with automatic library type detection (-l A), selective-alignment validation (--validateMappings), GC-bias correction (--gcBias), and sequence-specific bias correction (--seqBias). Salmon output files include transcript-level TPM values, estimated counts, and effective transcript lengths.
GEO
GSM9797113_r1