Post-Embryo FACS-sorted cells
tiling arrays (Waterston project, Miller subgroup)
As a control for the mRNA tagging experiments, RNA was isolated from young adult wild-type (N2) animal, and co-immunoprecipitation with anti-FLAG antibody to detect background signal. This was amplified for application as a labeled, double stranded cDNA to the Affymetrix C. elegans 1.0 whole genome tiling array. A smoothed density plot (log 2 transformed with a window size of 110) of probe intensities and predicted transcriptionally active regions (TARs) are shown.
Our experiments are designed to detect all C. elegans transcripts by hybridizing RNA to commerically available genome tiling arrays. To maximize the chances of detecting rare transcripts with limited expression in specific cells, we are extracting RNA from selected embryonic cells isolated by FACS and from postembryonic cells by use of the mRNA tagging method.
- Growth and isolation: Isolation of cell specific RNA by mRNA tagging, Worm staging and isolation, Worm growth, Worm staging and isolation, Worm staging and isolation, Worm growth
- Sample preparation: CDNA_amplification_for_tiling_arrays, Affy_Hybridization_and_Scanning, CDNA_amplification_for_tiling_arrays
- Data Analysis: Tiling_Array_TAR_analysis, Tiling_Array_Normalization_and_Smoothing, Tiling_Array_Signal_Extraction, Tiling_Array_TAR_analysis
- Antibodies: EZview Red ANTI-FLAG® M2 Affinity Gel
- Arrays: Affymetrix GeneChip C. elegans tiling 1.0R array
- Animals/Lines: L3-L4-reference, L2-panneural, NC1598, NC1021, body wall muscle, L2-excretory_cell, L2-glr, L2-intestine, OS3991, SD1075, mid-L2 larva 20dC, SD1084, L3-L4-hypodermis, NC1668, YA-CEPsh, L2-GABA_neurons, YA-reference, NC1790, SD1241, NC1627, L2-coelomocytes, Young Adult 20dC 72hr post-L1, NC694, L3-L4-dop, L3-L4-PVD_OLL, NC1842, NC1700, L2-reference, L2-A-class, L3-L4 larva 20dC 22h 23dC 24hr post-L1, N2
- Other Protocols: Isolation of cell specific RNA by mRNA tagging, Isolation of cell specific RNA by mRNA tagging, Affy_Hybridization_and_Scanning