Rrna removal kit

The lncRNA expression in d5 and d10 cells were analyzed through high-throughput sequencing (Decode Genomics, China). In brief, total RNA was extracted from cells through Trizol reagent, and the RNA concentration, purity, and integrity were assessed and adjusted. Then, ribosomal RNA (rRNA) was removed by rRNA Removal Kit (Epicentre), and the remaining RNA was proposed to generate a sequencing library according to the methods as previously described [13 (link)]. Following, the library was sequenced on Illumina Hiseq X10 platform (Illumina) and the expression of lncRNAs was analyzed by Feature Counts.

RNA isolation, library construction, and sequencing were performed by Novogene Corporation. RNA extraction was performed according to the manufacturer’s instructions and was quantified using a Bioanalyzer 2100 (Agilent Technologies, CA, USA). The quality and quantity of RNA were assessed using an RNA Assay Kit and a fluorometer (Life Technologies, CA, USA). RNA was frozen and stored at −80 °C immediately after production.
A total quantity of 3 μg RNA per sample was used as the input material for the RNA sample preparations from three HFHSD pigs (number as 120, 138, 146) and three control pigs (number as 157, 159, 161). First, ribosomal RNA was removed using an rRNA Removal Kit (Epicentre, WI, USA), and the rRNA-depleted sample was cleaned. Subsequently, sequencing libraries were generated. To select cDNA fragments between 150–200 bp in length, the library fragments were purified with an appropriate system (Beckman Coulter, Beverly, USA). Subsequently, USER enzyme was incubated with the size-selected, adaptor-ligated cDNA samples. PCR was performed using Phusion High-Fidelity DNA polymerase, Universal PCR primers, and an Index Primer. Finally, the products were purified and library quality was assessed. After cluster generation, the libraries were sequenced on an Illumina Hiseq 2000 platform, and 100-bp paired-end reads were generated.

Total RNA was isolated using TRIzol reagent and then treated with DNase I (Invitrogen Corporation, Carlsbad, CA, USA) as demonstrated before [7 (link)]. Microarray analysis was performed on the Agilent Array platform. Briefly, mRNA was purified from 1 g of total RNA following removal of rRNA with an rRNA removal kit (Epicentre Biotechnologies, Madison, WI, USA). Next, each sample was amplified and transcribed into fluorescent cRNA along the total length of the transcripts without 3′ bias by utilizing a random priming method. The labeled cRNAs were hybridized onto the Human LncRNA Array v.2.0 (8960K; Arraystar Inc., Rockville, MD, USA). After extensively washing the slides, the Agilent Scanner G2505B was employed to scan the arrays. Agilent Feature Extraction Software v. 10.7.3.1 was employed to analyze the acquired array images. Then, quantile normalization and subsequent data processing were performed using the GeneSpring GX v11.5.1 software package (Agilent Technologies, Santa Clara, CA, USA). Hot map and hierarchical clustering were used to illustrate systematic variations in the differentially expressed lncRNAs and protein-coding RNAs among samples.