PC-AssayContainer ::= {
{
assay descr {
aid {
id 624150,
version 0
},
aid-source db {
name "867",
source-id str "Mulder_2012",
date std {
year 2012,
month 6,
day 24
}
},
name "Chromatin factor siRNA screen in epidermal stem cells",
description {
"Primary human epidermal stem cells (keratinocytes) were transfected
with a siRNA library targeting 332 known and putative chromatin factors.
These screens were performed in 5 conditions in triplicate. Results are
depicted as activity scores and Z-scores, respectively."
},
protocol {
"Cell culture and differentiation:",
"Primary normal human keratinocytes (oral lka, foreskin kc and km
strains), obtained with appropriate ethical consent, were cultured on feeders
as described42. Prior to induction of differentiation, cells were grown,
feeder-free, in Keratinocyte Serum Free Medium (KSFM containing 30 mug/ml
Bovine Pituitary Extract and 0.2 ng/ml EGF; Gibco) for 2-3 days. At ~70%
confluency cells were incubated in KSFM containing 10 muM AG1478
(Calbiochem), 200 ng/ml recombinant human BMP2/7 (R&D systems), both, or 10%
foetal bovine serum (PAA).",
"",
"siRNA nucleofection:",
"siRNA nucleofections were performed with the Amaxa 96-well shuttle
system (Lonza). Keratinocytes were grown in KSFM to ~70% confluency,
harvested and resuspended in cell line buffer SF. 2x105 cells were used for
each 20 mul transfection (program FF-113) with 1-2 muM siRNA duplexes. This
is equivalent to 5-10 nM siRNA in conventional liposome-based transfections.
Transfected cells were incubated at ambient temperature for 5-10 minutes and
subsequently resuspended in pre-warmed KSFM. Silencer Select siRNAs were used
(Ambion/Applied Biosystems).",
"",
"siRNA library:",
"We designed a custom library (Silencer Select product, purchased from
Ambion) targeting a comprehensive set of human genes encoding known or
putative chromatin-factors. We included factors containing any of the
following domains or functions: PHD, BROMO, CHROMO, PWWP, tandem BRCT, TUDOR,
BAH, MBT, SET (including DOT1L), JMJC, JMJN, PRMT, HAT, HDAC, SIRT, DNMT,
MBD, and SNF2 ATP-dependent remodelers. After manual curation for redundant
entries a final list of 332 chromatin-associated factors was obtained.",
"",
"siRNA screening and data processing:",
"We used passage 2 lka keratinocytes for the siRNA screens. Our
custom library of 332 siRNA pools (3 duplexes/pool) was plated in four
96-well plates. Following transfection, keratinocytes were manually dispensed
into twenty 96-well plates (8,000 cells/well) containing pre-warmed KSFM.
This allowed analysis of quadruplicate plates for each of the five treatment
groups (vehicle, AG1478, BMP2/7, AG1478+BMP2/7 and 10% serum). Medium was
refreshed the next day. 72 hours after transfection cells were differentiated
for 48 hours. Cells were fixed in 4% paraformaldehyde (10 min, RT), washed
and permeabilised in PBS+0.2% Triton X-100 (10 min, RT). Following blocking
(PBS+10%serum, 30 min, RT), cells were stained using Transglutaminase I
specific antibodies (1:2000 mouse monoclonal BC.1 in blocking buffer) for at
least 1 hour (RT). After three washes, cells were stained with IR800
anti-mouse secondary antibodies (1:2000, LiCor) and a DNA stain, DRAQ5
(1:2000, Biostatus Ltd), in blocking buffer for 1 hour at RT. Cells were
washed 3 times and a final volume of 100 mul TBS was added. Plates were
scanned and analysed using the LiCor Odessey system and software with
consistent settings throughout the whole screen.",
"",
"As a transfection efficiency dependent background control we included
two independent siRNAs targeting TG1. After scanning and quantification, this
background was subtracted and TG1 levels for each individual well were
normalised to DRAQ5 signal to give a measure of differentiation/cell for each
population of siRNA transfected cells. High data quality was ensured by
confirming high pearson-correlation coefficients (Pearson-correlation>0.95)
of each replicate versus the mean of the quadruplicates.",
"A Z-score was subsequently calculated for each population.",
" ",
"Where Chi is the background corrected normalised intensity of a
specific well, Alpha and delta are the mean and standard deviation of
background corrected normalised intensities of all test siRNAs on the plate,
respectively. This calculation effectively transforms the data (without
affecting its distribution) to an average of zero and a standard deviation of
one. This standardised format allowed us to compile the results of all
conditions and experiments into one dataset of ~6,600 quantitative
measurements. The three best replicates were used for hierarchical clustering
and network analysis."
},
xref {
{
xref pmid 22729083,
type pcit
}
},
results {
{
tid 1,
name "NCBI Gene ID",
description {
"NCBI Entrez Gene database id of intended siRNA target gene"
},
type string,
unit none,
annot gene-target-id
},
{
tid 2,
name "Gene Symbol",
description {
"Official symbol of target NCBI Gene Id"
},
type string,
unit none
},
{
tid 3,
name "vehicle_1",
description {
"Assay activity defininitions: 0= Z-score between 1 and -1; 25=
Z-score greater than 1 or -1; 50= Z-score greater than 1.5 or -1.5; 75=
Z-score greater than 2 or -2; 100= Z-score greater than 2.5 or -2.5"
},
type float,
unit none
},
{
tid 4,
name "vehicle_2",
description {
"Assay activity defininitions: 0= Z-score between 1 and -1; 25=
Z-score greater than 1 or -1; 50= Z-score greater than 1.5 or -1.5; 75=
Z-score greater than 2 or -2; 100= Z-score greater than 2.5 or -2.5"
},
type float,
unit none
},
{
tid 5,
name "vehicle_3",
description {
"Assay activity defininitions: 0= Z-score between 1 and -1; 25=
Z-score greater than 1 or -1; 50= Z-score greater than 1.5 or -1.5; 75=
Z-score greater than 2 or -2; 100= Z-score greater than 2.5 or -2.5"
},
type float,
unit none
},
{
tid 6,
name "AG1478_1",
description {
"Assay activity defininitions: 0= Z-score between 1 and -1; 25=
Z-score greater than 1 or -1; 50= Z-score greater than 1.5 or -1.5; 75=
Z-score greater than 2 or -2; 100= Z-score greater than 2.5 or -2.5"
},
type float,
unit none
},
{
tid 7,
name "AG1478_2",
description {
"Assay activity defininitions: 0= Z-score between 1 and -1; 25=
Z-score greater than 1 or -1; 50= Z-score greater than 1.5 or -1.5; 75=
Z-score greater than 2 or -2; 100= Z-score greater than 2.5 or -2.5"
},
type float,
unit none
},
{
tid 8,
name "AG1478_3",
description {
"Assay activity defininitions: 0= Z-score between 1 and -1; 25=
Z-score greater than 1 or -1; 50= Z-score greater than 1.5 or -1.5; 75=
Z-score greater than 2 or -2; 100= Z-score greater than 2.5 or -2.5"
},
type float,
unit none
},
{
tid 9,
name "BMP2/7_1",
description {
"Assay activity defininitions: 0= Z-score between 1 and -1; 25=
Z-score greater than 1 or -1; 50= Z-score greater than 1.5 or -1.5; 75=
Z-score greater than 2 or -2; 100= Z-score greater than 2.5 or -2.5"
},
type float,
unit none
},
{
tid 10,
name "BMP2/7_2",
description {
"Assay activity defininitions: 0= Z-score between 1 and -1; 25=
Z-score greater than 1 or -1; 50= Z-score greater than 1.5 or -1.5; 75=
Z-score greater than 2 or -2; 100= Z-score greater than 2.5 or -2.5"
},
type float,
unit none
},
{
tid 11,
name "BMP2/7_3",
description {
"Assay activity defininitions: 0= Z-score between 1 and -1; 25=
Z-score greater than 1 or -1; 50= Z-score greater than 1.5 or -1.5; 75=
Z-score greater than 2 or -2; 100= Z-score greater than 2.5 or -2.5"
},
type float,
unit none
},
{
tid 12,
name "AG1478+BMP2/7_1",
description {
"Assay activity defininitions: 0= Z-score between 1 and -1; 25=
Z-score greater than 1 or -1; 50= Z-score greater than 1.5 or -1.5; 75=
Z-score greater than 2 or -2; 100= Z-score greater than 2.5 or -2.5"
},
type float,
unit none
},
{
tid 13,
name "AG1478+BMP2/7_2",
description {
"Assay activity defininitions: 0= Z-score between 1 and -1; 25=
Z-score greater than 1 or -1; 50= Z-score greater than 1.5 or -1.5; 75=
Z-score greater than 2 or -2; 100= Z-score greater than 2.5 or -2.5"
},
type float,
unit none
},
{
tid 14,
name "AG1478+BMP2/7_3",
description {
"Assay activity defininitions: 0= Z-score between 1 and -1; 25=
Z-score greater than 1 or -1; 50= Z-score greater than 1.5 or -1.5; 75=
Z-score greater than 2 or -2; 100= Z-score greater than 2.5 or -2.5"
},
type float,
unit none
},
{
tid 15,
name "10% KFCS_1",
description {
"Assay activity defininitions: 0= Z-score between 1 and -1; 25=
Z-score greater than 1 or -1; 50= Z-score greater than 1.5 or -1.5; 75=
Z-score greater than 2 or -2; 100= Z-score greater than 2.5 or -2.5"
},
type float,
unit none
},
{
tid 16,
name "10% KFCS_2",
description {
"Assay activity defininitions: 0= Z-score between 1 and -1; 25=
Z-score greater than 1 or -1; 50= Z-score greater than 1.5 or -1.5; 75=
Z-score greater than 2 or -2; 100= Z-score greater than 2.5 or -2.5"
},
type float,
unit none
},
{
tid 17,
name "10% KFCS_3",
description {
"Assay activity defininitions: 0= Z-score between 1 and -1; 25=
Z-score greater than 1 or -1; 50= Z-score greater than 1.5 or -1.5; 75=
Z-score greater than 2 or -2; 100= Z-score greater than 2.5 or -2.5"
},
type float,
unit none
}
},
revision 0,
activity-outcome-method screening,
substance-type nucleotide,
project-category literature-author
}
}
}