Supplementary Data: MIAME checklist

Experiment Design:

*The goal of the experiment – one line maximum

Dissecting Self-Renewal in Stem Cells with RNA Interference

*A brief description of the experiment

We present an integrated approach to identify genetic mechanisms that control self-renewal in mouse embryonic stem (ES) cells. Short hairpin RNA (shRNA) techniques are employed to down regulate a set of gene-products whose expression patterns suggest self-renewal regulatory functions. We focus on transcriptional regulators and identify seven molecules whose shRNA-mediated depletion induces differentiation, including four whose roles in self-renewal had not been demonstrated. Perturbations of individual gene-products are combined with dynamic, global analyses of gene expression, as well as computational approaches to elucidate provisional regulatory interactions. Our studies underscore the regulatory complexity of fate decision processes in ES cells.

*Experimental factors

shRNA induced differentiation time courses:

We analyze transcriptome dynamics after down-regulating each of the 8 self-renewal regulators identified in the current studies. These are Nanog, Oct4, Sox2, Esrrb, Tbx3, Tcl1, Mm.343880 and Dppa4. Gene specific shRNA transduced GFP+ cells were FACS purified daily during a seven day interval and used to interrogate Affymetrix microarrays. Empty H1P vector served as a reference.

* Experimental design - relationships between samples, treatments,

extracts, labeling, and arrays

In addition to below, see accompanying file ivanova_et_al_microarrays.txt

shRNA induced differentiation time courses:

ES cells (cell line CCE) were transduced with shRNA vectors specific for Nanog, Oct4, Sox2, Esrrb, Tbx3, Tcl1, Mm.343880 and Dppa4 as well as reference vector H1P. 48 hours after transduction cells were re-plated at the density 0.3x106cells per 10 cm dish. Simultaneously, GFP+ cells were FACS purified and used as day 0 time point. Re-plated cells were maintained in the presence of LIF. GFP+ cells from each culture were FACS purified every day for 7 consecutive days to obtain 8 day time course (d0-d7). 0.2x106 cells were used to prepare hybridization probes. Total RNA was isolated using the TRIzol Reagent (Invitrogen). T7(dT)24 primer was used in cDNA synthesis to incorporate a T7 RNA polymerase promoter into the cDNA. Biotinylated cRNA probes were synthesized using the BioArray High-Yield Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY). Fifteen g of biotin-labeled cRNA was fragmented for 35min. at 94oC in fragmentation buffer (Affymetrix; Santa Clara, CA), and hybridized to Affymetrix MOE 430 A and B oligonucleotide microarrays.

*Quality control steps taken

No replicates or dye swaps.

2. Samples:

*Manipulation of biological samples and protocols used (e.g.,

growth conditions, treatments, separation techniques).

Described in Experimental Design

*Technical protocols for preparing the hybridization extract

(e.g., the RNA or DNA extraction and purification protocol), and labeling.

Described in Experimental Design

3. Hybridization procedures and parameters:

*The protocol and conditions used for hybridization, blocking and

washing, including any post-processing steps such as staining.

Chips were hybridized overnight at 45C.

Fluidics protocol:

ProtocolEukGE-WS2v4 (Affymetrix)

Wash A1 Recovery Mixes0

Wash A1 Temperature (C)25

Number of Wash A1 Cycles10

Mixes per Wash A1 Cycle2

Wash B Recovery Mixes0

Wash B Temperature (C)50

Number of Wash B Cycles4

Mixes per Wash B Cycle15

Stain Temperature (C)25

First Stain Time (seconds)600

Wash A2 Recovery Mixes0

Wash A2 Temperature (C)25

Number of Wash A2 Cycles10

Mixes per Wash A2 Cycle4

Second Stain Time (seconds)600

Third Stain Time (seconds)600

Wash A3 Recovery Mixes0

Wash A3 Temperature (C)30

Number of Wash A3 Cycles15

Mixes per Wash A3 Cycle4

Holding Temperature (C)25

Scanner protocol:

Pixel Size3

Filter570

Scan Temperature

Number of Scans2

Scanner TypeHP

Solutions:

2X Stain Buffer:

250 mls
12X MES / 41.7
5M NaCl / 92.5
10% Tween-20 / 2.5
Water / 112.8

SAPE Stain Solution (#1 and #3)

1200 ul
2X Stain Buffer / 600 ul
Water / 540 ul
Acetylated BSA (50mg/ml) / 48 ul
SAPE (1mg/ml) / 12 ul

Antibody Stain Solution (#2)

600 ul
2X Stain Buffer / 300 ul
Water / 266.4 ul
Acetylated BSA (50mg/ml) / 24 ul
Normal goat IgG (10mg/ml) / 6 ul
Biotinylated antibody (0.5mg/ml) / 3.6 ul

WASH A

1 L
20X SSPE / 300
10% Tween-20 / 1
Water / 698
5% Antifoam / 1

WASH B

1 L
12X MES / 83.3
5M NaCl / 5.2
10% Tween / 1
Water / 910.5

Hybridization Mix

PER RXN
cRNA / 15 microg
B2 / 5 ul
20X Euk Hyb Controls / 15 ul
Herring Sperm DNA / 3 ul
BSA / 3 ul
2X Hyb Buffer / 150 ul
Water / Up to 300 ul

4. Measurement data and specifications:

*Raw Data

*The normalized and summarized data

*Data extraction and processing protocols

** Image scanning hardware and software, and processing

procedures and parameters.

Affymetrix

Scanner protocol:

Pixel Size3

Filter570

Scan Temperature

Number of Scans2

Scanner TypeHP

**Normalization, transformation and data selection procedures

and parameters.

Affymetrix MAS5