Affymetrix GeneChip Mouse Genome 430A 2.0 [Mouse430A_2]

Yy1 as molecular link between neuregulin and transcriptional modulation of peripheral myelination.

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RNA isolated from sciatic nerve of postnatal day 21 mice with conditional ablation of YY1 in the SChwann cells

GENOTYPE

Transcription profiling by array of mouse sciatic nerve from wild type and YY1 conditional knock outs

RNA isolated from sciatic nerve of postnatal day 21 mice with conditional ablation of YY1 in the SChwann cells


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-Unknown

P-AFFY-2

Labeling of samples was carried out according to the manufacturer’s standard protocol

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-6139

A Cy5-labeled reference library was generated by 10 cycles of PCR using 5' Cy5-17.93D (/Cy5/ATCGTAGGCACCTGAAA) and 17.92_c18_A20 (AAAAAAAAAAAAAAAAAAAA/iSp18/ATTGATGGTGCCTACAG) using a 45nM pool containing equal amounts of all 225 reference oligonucleotides as template. [Reference oligos are complimentary to all spots on the At_Dm_Ce_1k_v1_g1_2 array and contain constant 5' and 3' flanking adaptors to mimic small RNA cDNAs; See Axtell and Bartel for details] Labeled PCR products were fractionated through a 6% denaturing polyacrylamide gel, enabling excision of the shorter Cy5-labeled strands. Samples were adjusted to 5 µM in water.

Phenol / Chloroform

Described in Mallory et al., Plant Cell 13:571-583 (2001).

P-MEXP-6142

Small RNAs from 50 ug of a total RNA sample were fractionated, sequentially ligated to 3' and 5' adapters, and reverse transcribed as described by (Lau et al. 2001. Science 294:858-862). First-stage PCR used oligos 17.92 and 17.93D (Lau et al. 2001. Science 294:858-862), and proceeded until amplifications were in linear stage (as determined by visualization of products from successive cycles); typically 17-19 cycles. A 1/100 dilution of this reaction was used as template in a labeling PCR using oligos 5' Cy3-17.93D (/5Cy3/ATCGTAGGCACCTGAAA) and 17.92_c18_A20 (AAAAAAAAAAAAAAAAAAAA/iSp18/ATTGATGGTGCCTACAG) for 10 cycles to create an asymmetric PCR product. Labeled PCR products were fractionated through a 6% denaturing polyacrylamide gel, enabling excision of the shorter Cy3-labeled strands. Samples were adjusted to 5 µM in water.

P-MEXP-6143

2 µl of 5 µM Cy3-labeled sample and 2 µl of 5 µM Cy5-labeled reference was added to 20 µl of hybridization buffer (3.5X SSC, 1% [m/v] BSA, 0.1% [m/v] SDS, 93 µg/ml salmon testes DNA, 187 µg/ml Escherichia coli tRNA, 37 µg/ml poly-adenine) for a final concentration of 0.417 µM each. After heating for 4 minutes at 85°C, samples were applied to arrays that had been pre-hybridized for 45 minutes in 3.5X SSC, 1% (m/v) BSA, 0.1% (m/v) SDS, rinsed with de-ionized water, and dried. Arrays were incubated at 57°C for 16 hrs, then washed for 5 minutes at 50°C in 2X SSC, 0.1% SDS, followed by 10 minutes at room temperature in 0.1X SSC, 0.1% SDS, and 3 x 1 minute at room temperature in 0.1X SSC.

Axon_GenePix4000B

10 µm per pixel, line average 2, and constant PMT gains for both 635 nm and 532 nm.

Greenhouse - Long Days

Long-day conditions at 18C. 16 hours light, 8 hours dark. Organs harvested when 50-60 days old.

Short Days - Seedlings

Growth at 18C in 8 hr light, 16 hr dark. Seedlings harvested 10 days after sowing.

Long Days - Seedlings

Growth at 18C for 16hr light, 8 hr dark. Seedlings harvested after 10 days.

Roots

Arabidopsis root RNA samples were derived from Col-0 roots harvested from 14 day old plants grown in constant light in liquid culture (1X Murashige {MS} salts + vitamins, 1% sucrose, 5 mM MES-KOH pH 5.7) shaking at 60 rpm in constant light at 22°C.

P-MTAB-17073

The total RNA from two individual animals of each genotype were extracted seperately and pooled in equal molar to create one biological sample.

Transcription profiling by array of mouse sciatic nerve from wild type and YY1 conditional knock outs

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