GSM9513291: Cis_2-IP; Mus musculus; OTHER
Total RNA was extracted from samples. Ribosomal RNA (rRNA) was depleted from the total RNA to enrich for mRNA and lncRNA. The resulting RNA was chemically fragmented.The fragmented RNA was divided into Input and IP fractions. The IP fraction was incubated with an anti-ac4C antibody for immunoprecipitation. After washing, the immunoprecipitated RNA was eluted.Both IP and Input RNA were used for strand-specific library construction and sequenced on an Illumina platform. Strand-specific RNA-seq libraries were constructed from both the immunoprecipitated (ac4C-IP) and Input RNA samples. The protocol involved: 1) RNA end repair and 5' phosphorylation, 2) 3' adapter ligation, 3) reverse transcription with dUTP incorporation for strand specificity, 4) 5' adapter ligation, 5) cDNA amplification by PCR, and 6) size selection and purification of the final libraries. Library quality was assessed by Agilent Bioanalyzer, and qualified libraries were sequenced on an Illumina platform (e.g., NovaSeq 6000) to generate paired-end reads.