title: N.crassa 2489 x 8820 4 days post-fertilization perithecial tissue, 2007 experiment
design: Perithecia picked 4 days after fertilizing a 2489 (female) strain with 8820 (male) conidia.   Sequenced at Univ British Columbia and prepared by their protocols for miRNA sequencing in 2007.    Construction of a Small RNA sequencing library using the Illumina involves the following steps:  1.  Total RNA (1 ug) is added to a ligation reaction containing the Illumina RNA 3' adapter ((RA3) and T4 RNA Ligase 2, truncated.  The RNA 3' adapter is specifically modified to target microRNAs and other small RNAs that have a 3' hydroxyl group resulting from enzymatic cleavage by Dicer or other RNA processing enzymes.  2. The enzymatic reaction is ended by the addition of Stop Solution.  3. RNA 5' adapter (RA5) and RNA ligase reaction mix is combined with 3'-adapter modified RNA from step 2 above for the addition of the 5' adapter to small RNA molecules.  4. Adapter modified RNA is reverse transcribed using Invitrogen Superscript II Reverse Transcriptase.  5. PCR is performed with reverse transcribed cDNA to enrich for constructs that contain adapter molecules on both ends.  PCR is performed with two primers (RP1 and RPIX) that anneal to the ends of the adapters.  A total of 11 cycles of PCR is performed.  The RPIX adapter includes a unique index sequence that enables multiplex sequencing.   6. Amplified cDNA is size fractionated on a TBE PAGE gel.  A band representing fragments ranging in size from 145 to 160 bp is excised from the gel.  The adapter sequences are represented as 118 bp of the sequencing library constructs.  7. The band representing small RNA library constructs is eluted from the gel by soaking el fragments in elution buffer on a rotator.  8. Gel fragments are removed by passing the eluate over a 5 um filter.  9. The Small RNA library is concentrated by ethanol precipitation.  10. The concentration of the amplified library is measured on a Qubit fluorometer.  14. An aliquot of the amplified library is run on a DNA High Sensitivity bioanalyzer chip to validate the approximate size range of the library.  15. The concentration of cluster forming units in the library is defined by qPCR using the Kapbiosystems KAPA Library Quant Kit.  When inserts are shorter than the length of a seqeuncing read, Illumina adapter sequence will be present at the 3' end of the read.  Illumina adapter sequences can be trimmed from the sequence read prior to analysis using the sequences below.  For single end reads, you only need to trim the adapter sequences at the end of read 1.  For paired end reads, you should trim both the read 1 and the read 2 adapter seqeunces.  The Illumina adapter sequence that follows short inserts at the end of read 1 for Illumina TruSeq Small RNA libraries is shown below.      primers: 5'-primer GUU CAG AGU UCU ACA GUC CGA CGA UC and 3'-primer  pUC GUA UGC CGU CUU CUG CUU GidT