Neb next ultra 2 kits

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).

Libraries were prepared using NEBNext Ultra II kits (NEB) following the manufacturer’s protocol. Following library preparation, ChIP samples were purified as described [30 (link)], while input samples were purified using a 2:1 ratio of AmpureXP (GE) beads to library. Following PCR amplification, all samples were purified using AMpure XP beads with 0.9:1 beads to PCR ratio. Samples were sequenced using the Illumina NextSeq 500 platform with paired ends. See S3 and S4 Tables for library statistics.