Cleantag small rna ligation kit

For skeletal muscle samples, following the injury time course, tibialis anterior muscles were dissected and snap frozen with liquid nitrogen. The Norgen Total RNA Purification Kit (catalog no. 17200) was used to extract RNA from 10 mg tissue for each sample. For heart samples, following the infection time course, hearts were dissected, embedded in OCT, and frozen in liquid nitrogen. RNA was extracted with Trizol (Invitrogen, catalog no. 15596026) and glycogen precipitation for a small fraction of each of the heart samples. RNA quality was assessed via High Sensitivity RNA ScreenTape Analysis (Agilent, catalog no. 5067-5579) and all samples had RNA integrity numbers greater than or equal to seven.
Small RNA sequencing was performed at the Genome Sequencing Facility of Greehey Children’s Cancer Research Institute at the University of Texas Health Science Center at San Antonio. Libraries were prepared using the TriLink CleanTag Small RNA Ligation kit (TriLink Biotechnologies). Libraries were sequenced with single-end 50× using a HiSeq2500 (Illumina).

Total RNA was isolated using the Total RNA Purification Kit (Norgen Biotek, Thorold, Ontario, Canada) and quantified using the Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA). RNA integrity was assessed using the 4200 Tapestation (Agilent Technologies, Santa Clara, CA). Isolated RNA was used to make libraries for both smRNA-sequencing and RNA-sequencing. SmRNA-sequencing libraries were prepared by the Genome Sequencing Facility of Greehey Children’s Cancer Research Institute at the University of Texas Health Science Center at San Antonio using the TriLink CleanTag Small RNA Ligation kit (TriLink Biotechnologies, San Diego, CA). Eight libraries were sequenced per lane with single-end 50 × on the HiSeq3000 platform. RNA-sequencing libraries (polyA +) were prepared at Cornell’s Transcriptional Regulation and Expression Facility (TREx) using NEB Next Ultra II kits (New England BioLabs, Ipswich MA). Paired-end sequencing was performed at 20 million reads/sample on the NextSeq500 platform (Illumina, San Diego, CA).