Truseq stranded mrna library kit

RNA CapSeq libraries were prepared using a combination of TruSeq® Stranded mRNA Library Kit (Illumina) and SureSelectXT kit with a SureSelectXT custom capture library targeting the region of 400 kb on the X chromosome from OGT to CXCR3. cDNA was made from RNA using the TruSeq® Stranded mRNA Library Kit (Illumina). 500-100 ng of total RNA was used to prepare libraries that were first PolyA-bead captured to enrich for mRNA, followed by stranded reverse transcription and chemical shearing to generate appropriate stranded ~175 bp length cDNA inserts. These cDNA inserts were end-repaired, adenylated, ligated to adapter oligos and then amplified with 5 cycles of PCR according to manufacturer’s instructions. After quantification, 750ng of each amplified DNA sample was hybridized overnight with the Capture Library. Following capture cleanup, each gDNA library was amplified with additional 16 cycles of PCR, which also tagged each sample with an index-specific barcode. Final products were quantified using the TapeStation 2200 and pooled for rapid mode sequencing on the Illumina. RNA CapSeq libraries were sequenced at the Broad Institute Genomic Services as 101bp paired-end reads on an Illumina HiSeq2000 platform.

Three pairs of P60–P70 Rbfox1 Nes-cKO and wildtype male littermates from different litters were sacrificed and hippocampi were dissected out in ice-cold HBSS. Hippocampi were lysed in Trizol (Invitrogen) using a Tissue Tearor and RNA was extracted according to the manufacturer’s instructions. 1ug of total RNA was used for polyA selection and library construction using the Tru-Seq mRNA Stranded Library Kit (Illumina). Isolated PolyA-plus RNA was converted to cDNA and subjected to 50 nt paired-end sequencing using the standard Illumina platform on a Hi-Seq 2000.