title: MDAMB231_short_siControl_0Gy_bio_rep3
design: 4.5 million cells in Dulbeccos Modified Eagles Medium (DMEM; Thermo Fisher Scientific, #41966029) supplemented with 10% FBS (Sigma-Aldrich, #F9665-500ML) were transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific, #13778030) with a non-targeting siRNA pool (Dharmacon, #D-001910-10-50). One-and-a-half days later, the cells were mock-irradiated (0 Gy). Forty-eight hours later, the cells were collected by scraping and the RNA extracted using RNeasy Mini Kit (QIAGEN).  Total RNA was shipped to Novogene in dry ice for Illumina sequencing on Novaseq 6000, paired-end 150 bases. At Novogene, mRNA was purified from total RNA using poly-dT oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount. The original fluorescence images obtained from high throughput sequencing platforms are transformed to short reads by base calling. These short reads are recorded in FASTQ format, which contains base information (reads) and corresponding sequencing quality information.The sequenced reads (raw reads) often contain low quality reads and adapters, which will affect the analysis quality. Therefore, it is necessary to filter the raw reads to get the clean reads. The filtering process is as follows: 1) Remove reads containing adapters. 2) Remove reads containing N > 10% (N represents bases that cannot be determined). 3) Remove reads containing > 50% low quality (Q score  5) bases.