GSM9701686: osteoclast, ctrl3, day3; Mus musculus; RNA-Seq
RNA was harvested using a total RNA extraction kit (Tiangen). 1.3 ug of total RNA was used for the construction of sequencing libraries. Total RNA was extracted using the appropriate kit based on sample type (Tiangen DP411, Tiangen DP762-T1C, Aidlab RN40 with CLB or CTAB, or TRIzol, etc.). RNA quality and integrity were assessed using a NanoDrop2000 and an Agilent 2100 Bioanalyzer / LabChip GX, with the following criteria: total amount ≥ 2 μg, concentration ≥ 40 ng/μl, OD260/280 1.7-2.5, OD260/230 0.5-2.5, RIN ≥ 7 (non-plant) or ≥ 6.5 (plant) (or RIN 6.0-6.4 with 28S/18S ≥ 1 and no baseline elevation). mRNA Capture Beads were equilibrated for 30 min on a four-dimensional rotator. mRNA was enriched by two rounds of binding: total RNA was mixed with the beads, denatured at 65°C, held at room temperature for 5 min, and magnetically separated; after washing, mRNA was eluted in Tris Buffer at 80°C. For the second binding, Beads Binding Buffer was added, incubated for 5 min at room temperature, washed, and then Frag/Prime Buffer was added. mRNA fragmentation was performed in a thermal cycler according to sample quality. First-strand cDNA was synthesized using reverse transcriptase in a thermal cycler, and second-strand cDNA synthesis was carried out in a metal bath, followed by purification with 1.8X VAHTS™ DNA Clean Beads and elution in Nuclease-free Water. After end repair and A-tailing, adapters containing index sequences were ligated. The ligation products were purified and size-selected by sequential bead-based steps: 0.9X beads, then 0.6X beads (supernatant retained), and finally 0.1X beads (beads retained), with 80% ethanol washes. PCR amplification was performed, and the PCR products were purified using beads. Library quality was checked on a Qsep-400 system, and only libraries rated A (or B with negligible effect) were used. Libraries were constructed with the VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina (Vazyme, NR604-02) and purified with VAHTS™ DNA Clean Beads (Vazyme, N411-03). The qualified libraries were sequenced on an Illumina NovaSeq X Plus platform using paired-end 150 bp reads.