GSM9251335: Molm13, IP, replicate 2; Homo sapiens; OTHER
Cells were washed with PBS and lysed directly in RNAzol (Molecular Research Center) without trypsinization (1 mL per 10 cm plate of adherent cells or per 5 × 10^6 suspension cells). Lysates were homogenized, clarified by centrifugation, and crude small RNA was precipitated with isopropanol and water. RNA was purified on Zymo Research spin columns using guanidine/ethanol washes, on-column Proteinase K digestion, and water elution. Purified RNA was further treated with StcE mucinase (Sigma) followed by an additional Zymo column cleanup. Small RNAs were fractionated by mixing RNA with adjusted RNA binding buffer (Zymo RNA binding buffer mixed with one volume of ethanol) and recovered from the flow-through on a second Zymo column._x000B_To obtain secreted fractions, cells were cultured to ~80% confluence, washed with PBS, and incubated in serum-free medium for 48 h. Conditioned medium was cleared by centrifugation and filtration, concentrated with a 100 kDa cutoff filter, and ultracentrifuged to obtain small EVs (sEVs). Exomeres were isolated from the supernatant by additional ultracentrifugation, and supermeres were recovered from the exomere supernatant by higher-speed ultracentrifugation. Crude sEVs were further purified by iodixanol (Sigma) density-gradient centrifugation, and pooled fractions containing sEVs were washed and pelleted in PBS. Total or small RNA (50 ng–2 µg) was 3' polyadenylated with end blocking and purified. Polyadenylated RNA was subjected to enhanced rPAL labeling, biotin enrichment on neutravidin beads, and sialidase-mediated release of sialoglycoRNA. Released RNA was reverse-transcribed using anchored oligo-dT primers with template switching, followed by PCR amplification with dual-indexed primers. Amplicons (200–500 bp) were purified by gel extraction and bead clean-up. Libraries were assessed by TapeStation, pooled by concentration, and sequenced on an Illumina NovaSeq X (single-end 100 bp, dual 8 bp indexes).