Neb small rna library kit

Approximately the same amount of joint tissue per donor was pulverised into a powder with a dismembranator (Mikro-S, Sartorius, Melsungen, Germany) under liquid nitrogen, and total RNA was extracted using a miRNeasy kit (Qiagen, Crawley, UK).
Total RNA was extracted from 500 ​μl serum using a RNeasy Serum kit (Qiagen, Crawley, UK) with DNase treatment (Qiagen, Crawley, UK). RNA integrity (RIN) was confirmed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). Ribosomal RNA (rRNA) was depleted using the Ribo-Zero™ rRNA Removal Kit (Epicentre, Madison, USA). Quality control prior to sequencing was performed by Qubit and Bioanalyzer (Agilient Technologies, Santa Clara, USA) analysis with RNA Pico chips and small RNA chips to measure RNA quantity, RNA integrity and calculate microRNA percentage. 100 ​ng of rRNA-depleted RNA per sample were submitted for library preparation using a NEB small RNA library kit (New England Biolabs (NEB), Ipswich, USA) with the addition of tobacco acid pyrophosphatase (Epicentre, Madison, USA). Pooled samples were size selected (120-300bp) and purified with Ampure beads (Agencourt, Beckman-Coulter, High-Wycombe, UK). Sequencing was undertaken on an Illumina HiSeq 2000 platform (Illumina, San Diego, USA) using 100 base paired-end reads.

Total RNA was isolated from equal weights of joints and 500 μl serum using miRNeasy or RNeasy Serum kits respectively with DNase treatment (all Qiagen, Crawley, UK) to remove residual gDNA. Total RNA integrity (RIN) was confirmed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). Ribosomal RNA was depleted using the Ribo-Zero™ rRNA Removal Kit (Epicentre, Madison, USA). From 41 samples 100 ng of rRNA-depleted RNA was submitted for library preparation using NEB small RNA library kit (New England Biolabs (NEB), Ipswich, USA). To reduce workflow bias we used tobacco acid pyrophosphatase (Epicentre, Madison, USA) to remove potential 5′ caps found on some snoRNAs. Samples were amplified for 15 cycles, mixed into 3 pools, and size selected. The size-selected material was purified with Ampure beads (Agencourt, Beckman-Coulter, High-Wycombe, UK). SnoRNA sequencing was undertaken on the Illumina HiSeq 2000 platform (Illumina, San Diego, USA) using 100 base paired-end reads.