title: GSM8827564: dCas13b_ROBO1 rep1; Homo sapiens; RNA-Seq
library_name: GSM8827564
library_construction_protocol: Total RNA was extracted from hESCs with AG RNAex Pro Reagent (Accurate Biology, AG21101), adhering to the manufacturer's protocol Total RNA worked as input for the RNA sample preparation. Sequencing libraries were generated using NEBNext Ultra Libraries Prep Kit for Illumina (NEB, USA, Catalog #: E7530L) following manufacturer's protocol and index codes were added to attribute sequences to each sample. Sepecifically, poly-T oligo-attached magnetic beads were used to purified mRNA from total RNA. Fragmentation was applied with divalent cations under elevated temperature in NEB Next First Strand Synthesis Reaction Buffer(5X). M-MuLV Reverse Transcriptase (RNase H) and random hexamer primers were used to create first strand cDNA. RNase H and DNA Polymerase I were then used to synthesise second strand cDNA. The remaining overhangs were transformed into blunt ends by polymerase and exonuclease reactions. To become ready for hybridization, NEB Next Adaptors with hairpin loop structures were ligated after the 3' ends of DNA fragments had been adenylated. Purification of the library fragments was done using the AMPure XP system (Beverly, USA) to select cDNA fragments that were preferably 370–420 bp in length. Subsequently, 3 μL USER Enzyme (NEB, USA) was employed with adaptor-ligated, size-selected cDNA at 37°C for 15 min, and then 5 min at 95 °C before PCR. After that, Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) primer were used to carry out PCR. Finally, the PCR products were purified using the AMPure XP system, and the Agilent 5400 system (Agilent USA) was used to evaluate the library quality and quantify the results using QPCR (1.5 nM). The qualified libraries were pooled and sequenced using PE150 technique on Illumina platforms in Novogene Bioinformatics Technology Co., Ltd (Beijing, China), according to effective library concentration and necessary data amount.