GSM1645601: untreated run0141_lane8_read1_index29=5187MID; Mus musculus; miRNA-Seq
1 µg of total RNA was prepared using the Illumina TruSeq small RNA kit as per the manufacturer’s recommendations except for the library size selection as described from 42-45 days old 4L;C*,  wild-type (WT)  and Isofagamine treated  4L;C* mouse brain. Briefly, the RNA molecules in the sample were ligated with different adapters at their 5’ and 3’ ends using T4 RNA Ligases 2 and 1 respectively. After reverse transcription from the 3’ adapter, RNAse H treatment and 2nd strand synthesis from the 5’ adapter, the double stranded cDNA molecules were amplified by PCR with primers tailed with Illumina flowcell specific sequences and one of 48 possible 6-base indexes to complete the library construction. After pooling samples with different index in an equimolar fashion, the fraction of the total RNA libraries containing the miRNAs was selected by agarose gel electrophoresis using a SageScience Pippin Prep system which can be programmed to specifically elute the library fragments with inserts of size 18-25 bases. After quantitation and sizing verification on an Agilent Bioanalyzer, the library pool was diluted to 18 nM and separated into individual molecules by hybridization on an Illumina HiSeq flowcell to a density of 850k/mm2, bridge amplified on an Illumina cBot cluster generation instrument and then sequenced on an Illumina HiSeq2000 for 50 cycles in a single direction. After demultiplexing of each library pool according to its barcode sequence, a minimum of 2M reads per sample was available for analysis of miRNA expression levels.