title: GSM9444042: BH_1, control; Blautia hominis; RNA-Seq
library_construction_protocol: Total RNA was extracted and assessed for integrity and purity. RNA degradation and contamination were evaluated on 1% agarose gels, and RNA integrity was determined using the RNA Nano 6000 Assay Kit on the Bioanalyzer 2100 system (Agilent Technologies, USA). For library preparation, mRNA was purified from total RNA using oligo-dT probes to remove rRNA. Fragmentation was performed with divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer (5×). First-strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase, followed by RNase H digestion to remove RNA. Second-strand cDNA synthesis was performed using DNA polymerase I with dUTP substituted for dTTP. Overhangs were converted to blunt ends by exonuclease/polymerase treatment, followed by 3′-end adenylation and ligation of adaptors containing hairpin loop structures. USER enzyme treatment was applied to degrade the uracil-containing second strand. cDNA fragments of approximately 370-420 bp were size-selected using the AMPure XP system (Beckman Coulter, USA). The resulting libraries were amplified using Phusion High-Fidelity DNA polymerase with universal and index primers, purified again with AMPure XP, and assessed for quality on the Agilent Bioanalyzer 2100 system. Cluster generation was performed on a cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina) following the manufacturer's protocol.