title: Temporal transcriptome analysis of the chicken embryo yolk sac
design: High-throughput Serial Analysis of Gene Expression (SAGE) was performed using SOLiD4 technology (Applied Biosystems) at The Center for Genomic Technologies at the Hebrew University of Jerusalem. RNAs were reverse transcribed and SAGE libraries were prepared using a SOLiD SAGE Kit with Barcoding Adaptor Module (Applied Biosystems) which allows multiple samples to be processed in parallel. The system generates a library of 27-bp “tags” for all the transcripts in a cell and provides a method for analyzing the genome-wide expression levels of poly(A) transcripts. Briefly, mRNAs were attached to poly(T) coupled magnetic beads, cDNAs were synthesized using SuperScriptR III Reverse Transcriptase and E. coli DNA polymerase, digested with a 4-bp cutter enzyme (NlaIII), and ligated to a barcode adaptor sequence (unique for each library) containing a recognition site for a restriction enzyme (EcoP15I) that cuts DNA 27 bp away. After restriction and separation from beads, the tags were ligated to a second adaptor. The 27-bp transcripts, surrounded by two adaptors, were pooled into a single multiplexed sequencing reaction.