in order to obtain more mirna profiling and eliminate strain - specific effects , we selected six domesticated silkworm strains ( q , qiufeng ; b , baiyu ; qb , qiufeng    baiyu ; and bq , baiyu    qiufeng , r1 , and j1 ) and employed next generation sequencing platform to determine the novel and conserved mirnas hidden in the posterior silk gland of silkworm . in addition , according to gene expression cluster analysis , the third day of fifth - instar larva ( v3 ) is a key time point for silk synthesis and rapid cell growth . thus , animals were collected from three stages : ( 1 ) stage 1 : fourth instar molting to day 2 of fifth instar from q , b , qb , and bq ; ( 2 ) stage 2 : fifth instar day 3 to day 8 before spinning also from q , b , qb , and bq ; and ( 3 ) entire stage : stages 1  +  2 from r1 and j1 ( fig .  1 ) . the healthy animals were reared at 25  c on a natural diet of clean mulberry leaves to the scheduled time . posterior silk glands were dissected daily in 0.7% nacl at low temperature , rinsed with depc - treated water , and promptly stored in liquid nitrogen . following the manufacturer 's instructions , posterior silk glands were collected , and total rna was extracted with trizol reagent ( invitrogen , carlsbad , ca , usa ) . after quantified and examined with the agilent 2100 bioanalyzer ( agilent technologies , palo alto , ca ) ( fig . 2 ) , total rna of the desired size range ( 1830  nt ) was size - fractionated on a 15% page gel and ligated with 5 rna adapter ( 5-guu - cagaguucuacaguccgacgauc-3 ) using t4 rna ligase . ligated rna was size - fractionated on a 15% agarose gel to obtain 4060  nt fraction . subsequently , the 3rna adapter ( 5-puc - guaugccgucuucugcuugidt-3 ; p , phosphate ; idt , inverted deoxythymidine ) was also ligated with t4 rna ligase . after size - fractionated on a 10% agarose gel , the 7090  nt fraction excised and was subjected to rt - pcr . after amplified for 15  cycles , the pcr products were separated on agarose gels . the rt - pcr products were sequenced on the illumina platform ( beijing genomics institute or bgi , shenzhen ) ,   and 35  nt small rna reads were generated . after removed low quality reads , contaminants , poly a , and adapter sequences with perl script ( table 1 ) , clean reads with the size range of 1830  nt were retrieved and submitted to ncbi 's gene expression omnibus ( geo ) with the accession number gse 56380 . in order to obtain more mirna profiling and eliminate strain - specific effects , we selected six domesticated silkworm strains ( q , qiufeng ; b , baiyu ; qb , qiufeng    baiyu ; and bq , baiyu    qiufeng , r1 , and j1 ) and employed next generation sequencing platform to determine the novel and conserved mirnas hidden in the posterior silk gland of silkworm . in addition , according to gene expression cluster analysis , the third day of fifth - instar larva ( v3 ) is a key time point for silk synthesis and rapid cell growth . thus , animals were collected from three stages : ( 1 ) stage 1 : fourth instar molting to day 2 of fifth instar from q , b , qb , and bq ; ( 2 ) stage 2 : fifth instar day 3 to day 8 before spinning also from q , b , qb , and bq ; and ( 3 ) entire stage : stages 1  +  2 from r1 and j1 ( fig .  1 ) . the healthy animals were reared at 25  c on a natural diet of clean mulberry leaves to the scheduled time . posterior silk glands were dissected daily in 0.7% nacl at low temperature , rinsed with depc - treated water , and promptly stored in liquid nitrogen . following the manufacturer 's instructions , posterior silk glands were collected , and total rna was extracted with trizol reagent ( invitrogen , carlsbad , ca , usa ) . after quantified and examined with the agilent 2100 bioanalyzer ( agilent technologies , palo alto , ca ) ( fig . 2 ) , total rna of the desired size range ( 1830  nt ) was size - fractionated on a 15% page gel and ligated with 5 rna adapter ( 5-guu - cagaguucuacaguccgacgauc-3 ) using t4 rna ligase . ligated rna was size - fractionated on a 15% agarose gel to obtain 4060  nt fraction . subsequently , the 3rna adapter ( 5-puc - guaugccgucuucugcuugidt-3 ; p , phosphate ; idt , inverted deoxythymidine ) was also ligated with t4 rna ligase . after size - fractionated on a 10% agarose gel , the 7090  nt fraction excised and was subjected to rt - pcr . after amplified for 15  cycles , the pcr products were separated on agarose gels . the rt - pcr products were sequenced on the illumina platform ( beijing genomics institute or bgi , shenzhen ) ,   and 35  nt small rna reads were generated . after removed low quality reads , contaminants , poly a , and adapter sequences with perl script ( table 1 ) , clean reads with the size range of 1830  nt were retrieved and submitted to ncbi 's gene expression omnibus ( geo ) with the accession number gse 56380 . combining results from deep sequencing , microaary assay and bioinformatics analysis , we identified 728 novel mirnas ( including 55 mirna / mirna * duplex and 709 bm - specific mirnas expressed in the posterior silk gland in the period of the fourth - instar molting to the fifth - instar ( day 8 before ) spinning . these findings pave a way for further functional elucidation of these mirnas and their targets in silk production .
no special studies have been focused on the microrna ( mirna ) in the fifth - instar posterior silk gland of bombyx mori . here , using next - generation sequencing , we acquired 93.2 million processed reads from 10 small rna libraries . in this paper 
 , we tried to thoroughly describe how our dataset generated from deep sequencing which was recently published in bmc genomics . 
 results showed that our findings are largely enriched silkworm mirna depository and may benefit us to reveal the mirna functions in the process of silk production .