GSM6256866: Zf_Ko_3_EW8; Danio rerio; OTHER
RNA was harvested using Rneasy mini kit (Qiagen). 2 ug of total RNA was used for the construction of sequencing libraries. We reverse transcribed 1 ug of total RNA with the partial P7 adapter (Illumina_4N_21T) and dNTPs with the addition of spiked-in azido-nucleotides (AzVTPs) at 5:1. We click-ligated the p5 adapter (IDT) to the 5′ end of the cDNA with CuAAC. The p5 adaptor contained a UMI. We then amplified the cDNA for 17 cycles with 5' and 3′ indexing primer and purified it on a 2% agarose gel by extracting amplicon from 200-300 base pairs. We pooled the libraries and sequenced single-end, 100 base-pair reads on a NovaSeq (Illumina).