title: GSM9461556: Colon; mouse gut metagenome; RNA-Seq
library_construction_protocol: For each intestinal region, adjacent cryosections corresponding to Visium and Visium+PAP samples were processed for bulk metatranscriptomic analysis. Total RNA was extracted using the NucleoSpin RNA Stool Kit (Takara Bio, 740130.50). To remove contaminating genomic DNA, DNase treatment was performed using a two-step protocol: an initial treatment with Baseline-ZERO DNase (1 μL; LGC Biosciences, DB0715K) followed by a second treatment with TURBO DNase (3 μL; Thermo Fisher Scientific, AM2238) in 10 μL of the supplied buffer. A 1 μL aliquot of RNA was diluted in 99 μL of nuclease-free water, incubated at 37 °C for 30 minutes, and then placed on ice. RNA cleanup was performed using the RNA Clean & Concentrator kit (Zymo Research, R1013), with elution in 10 μL of nuclease-free water. Stranded total RNA-seq libraries were prepared using the SMARTer Stranded Total RNA-Seq Kit v3 -Pico Input Mammalian (Takara Bio, 634485), following the manufacturer's protocol. The four libraries were pooled and sequenced with MiniSeq sequencer (Miniseq High Output Reagent Cartridge, 150 Cycles, Ilumina).