GSM9039937: M1, HAND1-knockout, ROSA26-DOX-HAND1, 72h with DOX, rep1; Homo sapiens; RNA-Seq
RNA was extracted from samples using a standardized extraction method, followed by stringent quality control (QC) of RNA samples, including three key assessments: 1. Concentration assessment: RNA concentration and total yield were quantified using Qubit 4.0 Fluorometer (Thermo Fisher Scientific). 2. Integrity assessment: RNA integrity was evaluated with an Agilent 2100 Bioanalyzer (Agilent Technologies) using the RNA Integrity Number (RIN) metric. 3. Purity assessment: Sample purity was verified via absorbance ratios (A260/A280 and A260/A230) measured by a NanoDrop spectrophotometer (Thermo Fisher Scientific). mRNA with polyA tails was enriched using Oligo(dT) magnetic beads, followed by fragmentation at an optimized temperature and duration based on the desired fragment size, using a metal ion solution.The fragmented mRNA was used as a template to synthesize the first strand of cDNA with random oligonucleotide primers in the M-MuLV reverse transcriptase system.After first-strand synthesis, second-strand synthesis was performed, followed by end repair and A-tailing.T4 DNA ligase was used to catalyze the ligation of double-stranded DNA sticky ends with short adapters.Adapter-ligated products were purified using AMPure XP beads, and cDNA fragments of 300–350 bp were selected to ensure fragment uniformity.PCR amplification was performed to add sample index tags, followed by additional purification using AMPure XP beads to obtain the final library. After library construction, the libraries were initially quantified and diluted, followed by fragment size distribution analysis using the Agilent 2100 Bioanalyzer DNA 1000 kit. Qualified libraries were then accurately quantified via qRT-PCR to determine the effective concentration and ensure quality. The prepared DNA nanoballs (DNBs) were loaded onto a patterned array chip, and sequencing was performed using combinatorial probe-anchor synthesis (cPAS) technology. Sequencing primers, anchor molecules, and fluorescent probes were polymerized on the DNBs. A high-resolution imaging system was used to capture, read, and identify optical signals, generating single-base sequence information. This process was repeated in cycles to obtain the full sequence data.