title: mature miRNA sequencing of human brain using PCR-based barcodes
design: a)PCR-based bar-coding  Ligation of 3’ adapter was conducted by incubating 1 ?g of total RNA from the desired samples with 10 pmoles of 3’adenyated oligonucleotide, 10% DMSO, 20 U of RNAseInhibitor (Enzymatics Y924L) and 300 U of T4 RNA ligase 2 truncated (Enzymatics), at 22?C for 1hr. Following incubation, 10 pmoles of 5’adapter was added alongside 8 ?M ATP (Enzymatics N207-10-L) and 20 U of T4 RNA Ligase 1 (Enzymatics L605L) and incubated at 20?C for 1 hr. A third of the reaction product was used for reverse transcription of the adapter-microRNA-adapter fragments using 25 pmoles of 3’adapter compatible reverse transcription primer, and 200 U of Superscript III (Invitrogen 18080-044) as previously described1 followed by incubating at 48?C for 30 min. Following reverse transcription, PCR components were added directly to the RT reaction mixture by adding 1 X HF Phusion buffer, 25 pmoles of each PCR primers pairs (PCR1 and PCR2 bar-coding primers), 250 uM dNTPs, and 1 U of Phusion hotstart DNA polymerase (NEB F-540L). The reaction was thermal cycled as follow; 98°C for 30 sec, followed by 12 cycles of 98°C for 10 sec, 60°C for 20 sec and 72°C for 20 sec, and a final incubation at 72°C for 5 min and then paused at 4°C. The PCR products were purified on denaturing PAGE twice, as detailed in the supplementary protocol using crush and soak extraction1. Each individually bar-coded miRNA library was quality controlled on an Agilent Bioanalyzer and on a Nanodrop spectrophotometer, and combined at equimolar concentration in one unique library containing all the bar-coded samples prior to sequencing on a single Illumina lane.  The 5' adaptor sequence:  AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTC  The 3' Adaptor sequence:  ACGGGCTAATATTTATCGGTGGAGC  The barcode sequence for each library:  ATCACG - human brain  CGATGT - human brain  TTAGGC - human brain  TGACCA - human brain  ACAGTG - human brain  GCCAAT - human brain  CAGATC - human brain  ACTTGA - human brain  GATCAG - human brain  TAGCTT - human brain  GGCTAC - human brain  CTTGTA - human brain  The general structure is:  5' adaptor - mature miRNA - 3' adaptor - barcode