array scanning and feature extraction protocol
P-AFFY-6 Affymetrix CEL analysis
Title: Affymetrix EukGE-WS2v4_450 Hybridization. Description:
log2, GCRMA normalized data
This is a modified version of Affymetrix 'GeneChip Eukaryotic Small Sample Target Labeling Assay Version II' protocol involving the following steps: 1. Reverse transcription of total RNA with oligo-dT containing the T7 promoter. 2. Transformation of single strand cDNA to double strand cDNA with E. coli DNA polymerase. RNAse H is added to degrade RNA.3. In vitro Transcription using Ambion MEGAscript Kit according to manufacturer's protocol and subsequent RNA clean-up with Qiagen RNeasy Kit. 4. Second round of cDNA synthesis (first and second strand).5.In Vitro Transcription to Synthesize Biotin-Modified rRNA according to Affymetrix's protocol. (Parameters: Amplification = RNA polymerases, Mass unit = Micro gram)
Total RNA was extracted from 10000 GFP-positive cells with Arcturus PicoPure RNA Isolation Kit (Applied Biosystems) according to manufacturer's instructions. (Parameters: Extracted product = total_RNA, Amplification = none)
60 brains were dissociated into single cells and GFP-positive cells were isolated by Fluorescence Activated Cell Sorting (FACS).
Fly stocks were raised on standard cornmeal food at 25C and 40-50% relative humidity. (Parameters: time unit = seconds, temperature unit = C)