Name: GSM8228799
Instrument: Illumina NovaSeq 6000
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: PAIRED
Construction protocol: Both control group and dsRNA-injected embryos at 5 hpf were thoroughly lysed on ice using lysis buffer.The lysate was then centrifuged at 12000g at 4°C for 10 minutes to remove cellular components, including undigested material such as cell nuclei.Then, RNaseI was added to the supernatant and incubated at 25°C for 30 minutes to digest RNA, while the RNA fragments protected by ribosomes were preserved. Subsequently, an RNA inhibitor was added to terminate the digestion reaction. The lysate was subjected to sucrose density gradient centrifugation, and fractions containing single peaks at 260nm wavelength around the 80S monosome region were collected. The collected mixture was then thoroughly lysed using Trizol for RNA extraction. RNA fragments within the size range of 26-34nt was separated using polyacrylamide gel electrophoresis without RNase, and the corresponding bands were collected. The recovered bands were subjected to ligation reaction, where a preadenylylated and 3'-blocked linker (5' rApp-CTGTAGGCACCATCAAT-NH2 3') was attached to the 3' end of the RNA. The ligated products were then recovered by performing polyacrylamide gel electrophoresis. Subsequently, the recovered RNA fragments were reverse transcribed using a reverse primer to obtain extended cDNA molecules. The purified cDNA was subjected to circularization by using CircLigase (Epicentre, CL4111K), resulting in the formation of circular cDNA molecules. To remove residual rRNA components in the cDNA, a method involving hybridization with complementary primers specific to rRNA and subsequent heat denaturation was employed. The remaining circularized cDNA was then amplified through PCR, and barcode sequences were incorporated.