2 protocols
normalization data transformation protocol
The adapter was trimmed off of the 3’ end of each read. Reads were aligned to the human genome build 19 (hg19) using Bowtie version 0.12.3 allowing up to two mismatches and limiting the number of locations a read can map to the genome to 4 or fewer. The number of reads that overlap with the genomic locations of known mature microRNA from miRBase v16 and from the miRDeep predictions for miR- 4423 was counted using BEDTools v2.9.0.
nucleic acid library construction protocol
300ng of small molecular weight fraction (<200bp) RNA was pooled from individuals within each phenotype, never smoker (NS), current smoker (S), no cancer (NC) and cancer (C), for a total of 4 samples (900ng each). Each pooled sample was size fractioned using the Ambion flashPAGE fractionator to obtain small RNAs of 10-40nts. Small RNA sequencing libraries were created using SOLiD Small RNA Expression Kit (Ambion) starting with 200ng of the size fractionated small RNA following the manufacturer’s instructions. Libraries were sequenced on an Applied Biosystems SOLiD System to obtain 36 bp reads.