GSE133000
RNA sequence of mRNA in HUVEC cells after depleting EGFL6
Total RNA was isolated from each thymic sample using the standard TRIzol protocol (Invitrogen, Carlsbad, CA, USA). RNA quality was examined by gel electrophoresis and with a Nanodrop spectrophotometer (Thermo, Waltham, MA, USA). For RNA sequencing, RNA samples from 9 biological replicates were separated into three independent pools, each comprised of three distinct samples at equal amounts. Strand-specific libraries were constructed using the TruSeq RNA sample preparation kit (Illumina, San Diego, CA, USA), and sequencing was carried out using the Illumina HiSeq X Ten instrument by the commercial service of Genergy Biotechnology Co. Ltd. (Shanghai, China). The raw data was delieved into NCBI's Gene Expression Omnibius, and was handled by Perl and data quality was checked by FastQC v0.11.2. Clean reads were aligned to the chicken genome (release: Gallus gallus 4.0) from NCBI using Bowtie, with one mismatch allowed. As shown by previous study[2], the expression of the transcript was calculated by FPKM using Perl. Differentially expression transcripts (DETs) were determined using the MA-plot-based method with Random Sampling (MARS) model in the DEGseq package between different time points. The thresholds for determining DETs are P < 0.05 and absolute fold change = 2. Then DETs were chosen for function and signaling pathway enrichment analysis using GO and KEGG database. Overall design: 3 samples of control group and 3 samples of knock-down group