title: parental C4-2(endogenous ARRB1)_H3K4me3 ChIP

city: Cambridge

first: Chandra

last: Chilamakuri

address: Robinson Way

institute: Cancer Research UK Cambridge Institute

data_processing:
Single-end 36 bp reads generated by the Illumina HiSeq 2000 were aligned against the Human Reference Genome (assemby GRCh37, NCBI Build 37) using BWA version 0.5.9. Reads were filtered by removing those with a BWA alignment quality score less than 15 as well as duplicate reads.
Enriched regions of the genome were identified by comparing ChIP samples to input samples using MACS Version 1.4.2
Genome_build: GRCh37
Supplementary_files_format_and_content: MACS peaks in .txt file

source_name: C4-2_H3K4me3 ChIP

growth_protocol: RPMI & 10% Foetal Bovine Serum supplemented with G418 for the stable nucARRB1 lines

extract_protocol: Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.  Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.

treatment_protocol: no treatement

value: immortalised prostate epithelial cells

value: parental, endogenous levels of ARRB1

tag: factor

tag: cuture condition

value: untreated, cultured in RPMI+10%FBS

tag: chip antibody vendor

value: Diagenode

tag: chip antibody cat. #


title: nucARRB1 C4-2(high nuclear ARRB1)_H3K4me1 ChIP

status: Public on Nov 26 2014

type: SRA

country: United Kingdom

library_source: genomic

molecule: genomic DNA

organism: Homo sapiens

source_name: nucARRB1_H3K4me1 ChIP

tag: cell line

tag: cell type

tag: genotype/variation

value: stably expressing nucARRB1

tag: chip antibody


title: human prostate tissue_ARRB1 ChIP

source_name: prostate tissue_ARRB1 ChIP

tag: tissue

value: human prostate tissue

value: A1CT anti-ARRB1 gift from Pr R. Lefkowitz


title: human prostate tissue_H3K4me1 ChIP

source_name: prostate tissue_H3K4me1 ChIP


title: nucARRB1 C4-2(high nuclear ARRB1)_H3K4me3 ChIP

source_name: nucARRB1_H3K4me3 ChIP


title: nucARRB1 C4-2(high nuclear ARRB1)_ARRB1 ChIP

source_name: nucARRB1_ARRB1 ChIP


title: parental C4-2(endogenous ARRB1)_p300 ChIP

value: Santa Cruz


title: parental C4-2(endogenous ARRB1)_H3K4me1 ChIP

source_name: C4-2_H3K4me1 ChIP


title: nucARRB1 C4-2(high nuclear ARRB1)_RNAPolII ChIP

source_name: nucARRB1_RNAPolII ChIP

value: RNAPolII

value: anti-phosphoSer5 RNA pol II

value: AbCam


title: human prostate tissue_H3K4me3 ChIP

source_name: prostate tissue_H3K4me3 ChIP


title: parental C4-2(endogenous ARRB1)_RNAPolII ChIP

source_name: C4-2_RNAPolII ChIP


title: parental C4-2(endogenous ARRB1)_ARRB1 ChIP

source_name: C4-2_ARRB1 ChIP


title: PD88.2

city: Glasgow

first: Peter

middle: D.

last: Adams

address: Switchback Rd, Bearsden

institute: University of Glasgow, Beatson Institute for Cancer Research

description:
ribodepleted RNA
RNA-Seq of PD88 senescent IMR90 cells
Ribodepletion was performed using illumina epicentre Kit and libraires were prepared according to illumina protocol

library_source: transcriptomic

data_processing:
Basecalls performed using CASAVA 1.8.2 and filtered to only sequences passing the Illumina QC filter
Sequenced reads were mapped to hg18 whole genome using tophat 1.4.1 with parameters –solexa-quals, -p 12, --segment-length 18, -M, -G Homo_sapiens.NCBI36.54.gtf
Aligned reads normalised by library size (number of fragments), PCR duplicates were removed and subsequent reads piledup and converted to bigWig format
Genome_build: hg18
Supplementary_files_format_and_content: bigWig files represent pileups of the reads at each base.  bigWig files were normalised by library size and represent the number of reads at a given genomic location per million mapped reads and generated using UCSC wigToBigWig. bigWig files have PCR duplicates removed.

molecule: total RNA

source_name: IMR90 human fiboblasts

growth_protocol: Cells were grown using standard conditions and RNA harvested when plates were 80% confluent

extract_protocol:
RNA was extracted using RNA easy mini kit. Epicentre Ribodepletion kit was used to deplete ribosomal RNA.  Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 10 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: No treatment.

value: senescent


title: PD88.1


title: ER32.40HT.2

description: Ribodepleted RNA

extract_protocol:
RNA was extracted using RNA easy mini kit. Epicentre Ribodepletion kit was used to deplete ribosomal RNA.  Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 10 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols
Ribodepletion was performed using illumina epicentre Kit and libraires were prepared according to illumina protocol

treatment_protocol: ER32.40HT cells were treated with tamoxifen, control ER32 cells were treated with ethanol.  Cells were kept in treatment media.

value: ER32 + 40HT IMR90

tag: treatment

value: tamoxifen


title: ER32.1

description: Ribodepleted RNA

value: ER32 IMR9

value: ethanol


title: ER32.2

description: Ribodepleted RNA

value: ER32 IMR90


title: PD32.2

description:
ribodepleted RNA
RNA-Seq of PD32 proliferating IMR90 cells
Ribodepletion was performed using illumina epicentre Kit and libraires were prepared according to illumina protocol

value: proliferating


title: ER32.40HT.1

description: Ribodepleted RNA


title: PD32.1


title: H3.3 Senes Input DNA

description: corresponding input DNA

data_processing:
Basecalls performed using CASAVA 1.8.2
Sequenced reads were trimmed using trim galore v0.3.0 with parameter --quality 20 and remaining default settings
Sequenced reads were mapped to hg19 whole genome using bowtie v2.1.0 with parameter -p 15 and remaining default settings
Multimapped reads were removed using samtools 0.1.16 and duplicate reads removed using picard tools v1.98 mark duplicates
Aligned reads normalised by library size, extended to 150bp and converted to bigWig format
USeq 8.6.0 was used to perform HIRA peak finding (normalised to input) with the following steps. BED files converted to Useq Point Format (Tag2Point). Satellite Repeat regions were removed from the dataset (FilterPointData). ScanSeqs used to identify enriched windows (-w 200 -p 150 -f). Enriched Regions Extracted (EnrichedRegionMaker) with 5% FDR and 2 fold change (-i 2,4 -s 13,1)
SICER v1.1 was used to perform histone peak finding (H3.3 was normalised to input, H4K16ac to H4) using redundancy threshold of 1, window size of 200bp, fragment size of 150bp, effective genome fraction of 0.75, gap size of 200bp and R of 0.01.
Differential binding sites for H4K16ac were identified using Diffbind v1.8.3 with the following steps. Reads were counted using an insert length of 150bp and scored using DBA_SCORE_READS_MINUS. Enriched regions were identified using the full library size, DBA_DESEQ method and remaining defaults.
Genome_build: hg19
Supplementary_files_format_and_content: BigWig files were normalised by library size and represent the number of reads at a given genomic location per million mapped reads and generated using UCSC wigToBigWig.  peak files were generated with USeq and thresholded at 5% FDR and 2 fold change cutoffs

source_name: Fibroblasts

growth_protocol: IMR90 cells were cultured in DMEM supplemented with 20% (v/v) FBS, 2 mM L-Glutamine, 50 U/ml Pen-Strep and incubated at 37°C in a humidified 5% CO2 and 3% O2 atmosphere. Every 4-5 days the cells were passaged to maintain 40%–90% confluence level until they stopped proliferating through Replicative senescence or oncogene induced senescence

extract_protocol:
Cross-linked cells were solubilized by ultrasound treatment (Bioruptor Diagenode) to generate DNA fragmentation in 150-300 bp range. Protein-DNA complexes were isolated using corresponding antibody pre-immobilized on Dynabeads (Invitrogen).
Libraries were prepared according to NEB's instructions accompanying the NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina®(E6240S). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were isolated using Agencourt AMPure XP beads (Beckman Coulter) and PCR-amplified using Illumina primers. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.

treatment_protocol: Cells were harvested by trypsinization and precipitated by centrifugation. Cells pellets were resuspended in 1% formaldehyde in DMEM and incubated for 5 more minutes. Cross-linking was quenched by adding 1/10 of volume of 1.25 M glycine (125mM final) and incubating for 5 min at room temperature. Media was aspirated and cells were rinsed twice with PBS, scraped into ice-cold PBS supplemented with protease inhibitors (Sigma, P8340) and collected by centrifugation (5 min at 200g, 4°C). Cell pellets were snap frozen in dry ice with ethanol and kept at -70°C before use. For HIRA ChIP IMR90 cells were grown to 80%–90% confluency in 15 cm plates. Three plates (about 20 million cells) were washed with phosphate buffered saline (PBS) and scraped into 6 ml of 10 mM Tris, 30 mM NaCl, 0.1% (v/v) NP40, 3 mM MgCl2 pH 7.5 supplemented with protease (Sigma, P8340) and phosphatase (Sigma, P5726) inhibitor cocktails. Following 10 min incubation on ice the suspension was centrifuged (3 min at 300 g, 4°C) to produce nuclear pellet. This pellet was resuspended in 1ml of benzonase buffer (50 mM Tris pH 7.5, 300 mM NaCl, 0.5% (v/v) NP40, 2.5 mM MgCl2) with protease and phosphatase inhibitors followed by addition of 125 U of benzonase (Sigma, E1014). The lysate was thoroughly mixed and incubated on ice for 30 min. Supernatant was recovered by centrifugation (5 min at 13000 g, 4°C), separated from the pellet and diluted with 1 ml of dilution/stop digestion buffer (50 mM Tris pH 7.5, 100 mM NaCl, 0.5% (v/v) NP40, 15 mM EDTA, protease and phosphatase inhibitors). The resulting lysate was used as chromatin preparation for ChIP.

tag: cell state


title: d1060_48A

city: Tulln

first: Harald

last: Berger

state: NOE

address: Konrad-Lorenzstraße 43

department: DAGZ

country: Austria

institute: Universität f. Bodenkultur Wien

data_processing:
Mapping was performed using Novoalign (NovoCraft)
reverse transcripts were counted using python script HTSeq (Anders et al., 2014)
Normalization and statistics were done using R/Bioconductor and the limma and edgeR packages
mean-variance weighting (voom) and TMM normalization (Gentleman et al., 2004)
Genome_build: A_nidulans_FGSC_A4_version_s10-m03-r07
Supplementary_files_format_and_content: [eset4ncbi.txt] Tab-delimited file, normalized log2(CPM) counts per million reads, rows for each gene, columns for samples WT_17A, WT_17B, WT_48A, WT_48B, d1060_17A, d1060_17B, d1060_48A, d1060_48B
Supplementary_files_format_and_content: [*count.txt] raw counts

organism: Aspergillus nidulans

source_name: kdmA 48 h growth

growth_protocol: ANM minimal media, complete medium, supplements and growth conditions were as described by Todd et al., 2007.

extract_protocol:
Total RNA was isolated from filtered, flash frozen mycelia using a Fastprep bead-beater (MP Biomedicals) and Trizol reagent (Invitrogen) following the manufacturers’ protocol.
Illumina sequencing libraries were made from RNA samples according to TruSeq RNA Sample prep kit v2 (Illumina) following the manufacturers protocol with 1µg total RNA input. 50 bp single end sequencing was performed using a HiSeq Illumina sequencer. Obtained sequences were de-multiplexed, quality controlled and mapped on the Aspergillus nidulans genome assembly (A_nidulans_FGSC_A4_version_s10-m03-r07).

treatment_protocol: Two different growth periods were applied, 17h and 48h.

tag: strain

value: kdmA

tag: growth period

value: mycelia


title: d1060_48B


title: WT_48B

source_name: wild type 48 h growth

value: wild type


title: d1060_17A

source_name: kdmA 17 h growth


title: d1060_17B


title: WT_17B

source_name: wild type 17 h growth


title: WT_17A


title: nucARRB1 C4-2(high nuclear ARRB1)_p300 ChIP

source_name: nucARRB1_p300 ChIP

value: immortalised prostate epithelial cells

value: stably expressing nucARRB1

value: untreated, cultured in RPMI+10%FBS


title: Lito_infected_rep1

status: Public on Nov 25 2014

city: Edinburgh

first: Alasdair

last: Ivens

address: Kings Buildings

institute: Centre for Immunity, Infection and Evolution

data_processing:
Primers were trimmed from the raw fastq format sequence data using cutadapt software (v1.3; parameters -O 6 -m 16 -q 20).
Only trimmed reads that contained the adapter, were 16-40 nt in length and were present at 2 or more copies per sample were retained.
Simple scripts were used to generate non-redundant sequence sets (collapsed) in fasta format from each sample.
Genome_build: http://badger.bio.ed.ac.uk/filarial/fileDownload/zip_download?fileName=Litomosoides_sigmodontis_2.1.fna
Supplementary_files_format_and_content: Processed data files contain the non-redundant sequences from each sample.

organism: Litomosoides sigmodontis

source_name: Mouse serum

growth_protocol: Six week-old  BALB/c mice were infected with Litomosoides sigmodontis (subcutaneous inoculation of 40 L3).

extract_protocol:
200 uL of serum was extracted with miRNA easy kit (Qiagen) and libraries generated following the TruSeq protocol
TruSeq Small RNA kit

treatment_protocol: Gel-separated serum (BD Microtainer) was collected by arterial ex-sanguination at 60 days post infection, which was confirmed by detection of adult worms in the mouse pleural cavity and microfilariae in peripheral blood

tag: host organism

value: BALB/c mouse

tag: host tissue

value: serum

tag: replicate

tag: infection status

value: infected


title: Cameraria ohridella

city: Oxford

middle: W. H.

last: Holland

address: South Parks Road

department: Department of Zoology

institute: University of Oxford

data_processing:
Bowtie index created for each of the Cameraria ohridella and Pararge aegeria genome assemblies
miRDeep2 "mapper.pl" script used to parse fastq to fasta, clip adapters, remove entries with non-canonical letters, collapse reads and map reads to bowtie index
Size selection done on collapsed reads to exclude sequences outside the range of 18-24 nt
miRDeep2 "mirdeep2.pl" script used to predict miRNA hairpins based on processed small RNA reads
Predicted hairpins filtered based on structure and free energy as well as comparisons to existing miRNA data
Genome_build: Cameraria ohridella and Pararge aegeria genome assemblies available in the Oxford Research Data Archive (doi 10.5287/bodleiandury.3)
Supplementary_files_format_and_content: Tab-delimited text files indicating each predicted miRNA precursor sequence as well as sequence and read counts for each of its mature products

organism: Cameraria ohridella

source_name: Cameraria ohridella whole body

extract_protocol:
TRIzol extraction followed by DNase I digestion
Library construction performed by the Beijing Genomics Institute

tag: developmental stage

value: Larva, pupa, adult

tag: growth

value: Wild caught

value: whole body


title: Lito_infected_rep2


title: WT_48A


title: Rpb1 WT RASL-seq Rep2

city: Aurora

first: David

middle: L.

last: Bentley

state: CO

department: Biochemistry and Mol. Genetics

country: USA

institute: U. Colorado School of Medicine

description:
polyA+ RNA
RASL- seq of polyA+ RNA

data_processing:
Library strategy: RASL-Seq
RNA-Seq reads were aligned using TophHat v1.4.1 (Pair-ends were treated as single-ends)
GRO-Seq reads were aligned using Bowtie v0.12.7
Log2 differential expressions of RNA-Seq were calculated using edgeR v3.7.17
profiles of Gro-Seq were calculated using 50bp bin counting
Genome_build: GRCh37/hg19 assembly
Supplementary_files_format_and_content: GRO-Seq: bigWig
Supplementary_files_format_and_content: RASL-Seq and RNA-Seq: bigBed (log2 fold change at 7th column)

molecule: polyA RNA

source_name: HEK293 Flp-in T-REx pcDNA5 Rpb1 Amr WT

growth_protocol: Cells were grown in DMEM 10% FCS, 200μg/ml hygromycin B, 6.5μg/ml blasticidin, and pen/strep

extract_protocol:
RNA-easy (Qiagen)
Illumina TruSEQ RNA Library Kit v2. Cat # RS-122-2001 from polyA+ RNA with random priming.
Paired-end (100 bp) RNA-seq reads from biological replicate samples  were mapped individually without merging by Tophat (v1.4.1) with a default option.

treatment_protocol:
DMEM 10% FCS,+hygromycin+blasticidin+doxycycline+a-amanitin
Amr Rpb1 was induced with doxycycline (2.0 mg/ml)  for 12-16 hr then treated with a-amanitin (2.5 mg/ml) for a further 42 hr before harvesting  RNA for RNA-seq and RASL-seq. For GRO-seq cells were doxycyline induced and a-amanitin treated as above then treated with DRB (100 mM) for 3 hr to stall pol II at start sites. Controls had no DRB added. DRB was washed out by replacing the medium and cells harvested at 10 and 20 minute time points. Nuclei were prepared for each sample and GRO-seq with Br-UTP labelling was performed as in Core et al Science, 322: 1845, 2008.

value: doxycycline induced, a-amanitin (2.5 mg/ml) 42 hr


title: Holy L3

data_processing:
Primers were trimmed from the raw fastq format sequence data using cutadapt software (v1.3; parameters -O 6 -m 16 -q 20).
Only trimmed reads that contained the adapter, were 16-40 nt in length and were present at 2 or more copies per sample were retained.
Simple scripts were used to generate non-redundant sequence sets (collapsed) in fasta format from each sample.
Genome_build: http://www.nematodes.org/genomes/heligmosomoides_polygyrus/nHp_1_0.fna.gz
Supplementary_files_format_and_content: Processed data files contain the non-redundant sequences from each sample.

organism: Heligmosomoides polygyrus

source_name: L3 stage worms

growth_protocol: For collection of H. polygyrus secretion product, CF1 mice are infected with infective-stage larvae by gavage and adult parasites collected from the small intestine 14 days post infection. The worms are maintained in serum-free media in vitro.

extract_protocol:
Total RNA was size selected on 15% denaturing PAGE and libraries prepared from the 18-30 nt fraction using Illumina Small RNA preparation kit version 1.5 and sequenced on the GAIIX, the Hpoly HES sample was pooled with RNA prepared in parallel from 60-90 size selection. For analysis of vesicle and non-vesicle fractions, small RNA libraries were prepared using the TruSeq kit and sequenced on MiSeq platform.
Illumina Small RNA preparation kit v1.5 and TrueSeq Small RNA kit

treatment_protocol: For separation of the vesicle and non-vesicle fractions, filtered secretion produce is ultracentrifuged at 100,000 g for 2 hours followed by 2 washes in PBS.

tag: sample type

value: L3 stage worms

tag: kit

value: Illumina Small RNA preparation kit v1.5


title: Hpoly Eggs

source_name: Worm eggs

value: Worm eggs


title: R10

source_name: Sup fraction rep 1

value: Supernatant fraction

value: TrueSeq small RNA preparation kit


title: Hpoly HES

source_name: Secreted HES product

value: Secreted HES product


title: R11

source_name: Sup fraction rep 2


title: R12

source_name: Sup fraction rep 3


title: Hpoly Adult

source_name: Adult worms

value: Adult worms


title: Rpb1 E1126G RASL-seq Rep3

description:
polyA+ RNA
RASL- seq of polyA+ RNA
supplementary file: exons.rs_WT_vs_E1126g.dj.gz

source_name: HEK293 Flp-in T-REx pcDNA5 Rpb1 Amr E1126G

tag: cell line


title: Rpb1 E1126G RASL-seq Rep2


title: Rpb1 E1126G RASL-seq Rep1


title: Rpb1 WT RASL-seq Rep1

description:
polyA+ RNA
RASL- seq of polyA+ RNA


title: Rpb1 H1106Y RASL-seq Rep1

description:
polyA+ RNA
RASL- seq of polyA+ RNA
supplementary file: exons.rs_WT_vs_H1106Y.dj.gz

source_name: HEK293 Flp-in T-REx pcDNA5 Rpb1 Amr H1106Y


title: Rpb1 H1106Y RASL-seq Rep2


title: Rpb1 H1106Y RASL-seq Rep3


title: Rpb1 R749H RASL-seq Rep3

description:
polyA+ RNA
RASL- seq of polyA+ RNA
supplementary file: exons.rs_WT_vs_C4.dj.gz

source_name: HEK293 Flp-in T-REx pcDNA5 Rpb1 Amr R749H


title: Rpb1 R749H RASL-seq Rep2


title: dcl3 replicate 2 small RNA

city: Dallas

first: Hume

last: Stroud

state: TX

address: 5323 Harry Hines Blvd

institute: UT Southwestern

data_processing:
First adapter sequences were removed. Reads were mapped to the TAIR10 genome using Bowtie by allowing no mismatches and only keeping reads that uniquely map to the genome. Small RNA   counts were normalized to the size of each small RNA library by dividing to the number of reads to the number of total uniquely mapping reads of 18-34 bp in size.
Genome_build: TAIR10
Supplementary_files_format_and_content: read counts of small RNA sequences

organism: Arabidopsis thaliana

source_name: Inflorescence tissue containing young flowers_dcl3

growth_protocol: Plants were grown in the greenhouse at 16h light, 8h dark cycles.

extract_protocol:
Total RNA was isolated with TRIzol (Ambion) from 0.1 g flowers. High-molecular-weight RNA was precipitated with 12.5% PEG8000 and 0.5 M NaCl. Low-molecular-weight RNA from the supernatant was ethanol-precipitated and 15-40-nt RNAs were excised from denaturing polyacrylamide/Urea gels and purified in final 6 µl water.
Individual TruSeq small RNA libraries from three biological replicates each were generated according to the manufacturer’s instructions (Illumina), except that 13 cycles of PCR amplification were used.

tag: genotype

value: inflorescence


title: dcl3 replicate 3 small RNA


title: drm1/2 replicate 2 small RNA

source_name: Inflorescence tissue containing young flowers_drm1/2


title: dcl3 replicate 1 small RNA


title: drm1/2 replicate 3 small RNA


title: drm1/2 replicate 1 small RNA


title: Rpb1 E1126G GRO-seq washout 20'

description:
Br-UTP labeled nascent RNA
Gro-seq of nuclear runon transcripts

data_processing:
Library strategy: GRO-Seq
RNA-Seq reads were aligned using TophHat v1.4.1 (Pair-ends were treated as single-ends)
GRO-Seq reads were aligned using Bowtie v0.12.7
Log2 differential expressions of RNA-Seq were calculated using edgeR v3.7.17
profiles of Gro-Seq were calculated using 50bp bin counting
Genome_build: GRCh37/hg19 assembly
Supplementary_files_format_and_content: GRO-Seq: bigWig
Supplementary_files_format_and_content: RASL-Seq and RNA-Seq: bigBed (log2 fold change at 7th column)

treatment_protocol:
DMEM 10% FCS,+hygromycin+blasticidin+doxycycline+a-amanitin+DRB washout
Amr Rpb1 was induced with doxycycline (2.0 mg/ml)  for 12-16 hr then treated with a-amanitin (2.5 mg/ml) for a further 42 hr before harvesting  RNA for RNA-seq and RASL-seq. For GRO-seq cells were doxycyline induced and a-amanitin treated as above then treated with DRB (100 mM) for 3 hr to stall pol II at start sites. Controls had no DRB added. DRB was washed out by replacing the medium and cells harvested at 10 and 20 minute time points. Nuclei were prepared for each sample and GRO-seq with Br-UTP labelling was performed as in Core et al Science, 322: 1845, 2008.

value: DRB washout t=20min.


title: suvr2 replicate 3 small RNA

source_name: Inflorescence tissue containing young flowers_suvr2


title: Rpb1 E1126G GRO-seq washout 10'

value: DRB washout t=10min.


title: suvr2 replicate 2 small RNA


title: suvr2 replicate 1 small RNA


title: frg1to5 replicate 2 small RNA

source_name: Inflorescence tissue containing young flowers_frg1to5

value: frg1 to frg5


title: frg1to5 replicate 1 small RNA


title: frg1to5 replicate 3 small RNA


title: Rpb1 H1106Y GRO-seq  DRB3hr

treatment_protocol:
DMEM 10% FCS,+hygromycin+blasticidin+doxycycline+a-amanitin+DRB
Amr Rpb1 was induced with doxycycline (2.0 mg/ml)  for 12-16 hr then treated with a-amanitin (2.5 mg/ml) for a further 42 hr before harvesting  RNA for RNA-seq and RASL-seq. For GRO-seq cells were doxycyline induced and a-amanitin treated as above then treated with DRB (100 mM) for 3 hr to stall pol II at start sites. Controls had no DRB added. DRB was washed out by replacing the medium and cells harvested at 10 and 20 minute time points. Nuclei were prepared for each sample and GRO-seq with Br-UTP labelling was performed as in Core et al Science, 322: 1845, 2008.

value: DRB (100 mM) 3hr


title: Rpb1 H1106Y GRO-seq washout 20'


title: Rpb1 H1106Y GRO-seq washout 10'


title: Rpb1 E1126G GRO-seq control


title: Rpb1 R749H RASL-seq Rep1


title: frg1/2 replicate 2 small RNA

source_name: Inflorescence tissue containing young flowers_frg1/2


title: frg1/2 replicate 1 small RNA


title: Col0 replicate 2 small RNA

source_name: Inflorescence tissue containing young flowers_Col1

tag: cultivar

value: wildtype


title: Rpb1  R749H GRO-seq DRB3hr


title: Rpb1  R749H GRO-seq control


title: Rpb1  R749H GRO-seq washout 10'


title: Col0 replicate 3 small RNA

source_name: Inflorescence tissue containing young flowers_Col2


title: Rpb1 WT GRO-seq washout 20'

source_name: HEK293 Flp-in T-REx pcDNA5 Rpb1 Amr WT


title: frg1/2 replicate 3 small RNA


title: Rpb1 WT GRO-seq washout 10'


title: Col0 replicate 1 small RNA

source_name: Inflorescence tissue containing young flowers_Col0


title: frg1 frg2 replicate 2 mRNA

data_processing:
Reads (fastq format) were mapped to the TAIR10 genome with tophat-2.0.12 and bowtie-1.0.0 using parameters -i 40 -I 1000 -g 1 -N 1 --bowtie1 --bowtie-n --library-type fr-unstranded --no-coverage-search --segment-mismatches 1 --segment-length 25 --min-segment-intron 40 --max-segment-intron 1000.
bedGraph files were generated from bam files using bedtools2.
Genome_build: TAIR10
Supplementary_files_format_and_content: bedGraph files indicate the number and position of uniquely mapping reads (column1=chromosome, column2=start, column3=end, column4=count).

source_name: Aerial tissue from 14 days old seedlings_frg1/2

growth_protocol: Plants were grown on 1x MS medium at 21 ºC and continuous light for 14 days after germination.

extract_protocol:
Total RNA was isolated with TRIzol (Ambion) from 0.1 g of tissue following the product manual. 2 µg of total RNA were treated with DNAse I, amplification grade (Invitrogen) for 15 min at room temperature. DNAse I was heat inactivated with 2.5 mM EDTA at 65 ºC for 10 min.
Individual libraries for each biological replicate were generated from 2 µg total RNA according to the manufacturer’s instructions (Illumina).

tag: age

value: 14 days old

value: aerial tissue


title: Rpb1 WT GRO-seq DRB3hr


title: Rpb1 E1126G GRO-seq  DRB3hr


title: frg1 frg2 replicate 1 mRNA

tag: tissue


title: Rpb1 WT GRO-seq control

tag: treatment


title: Rpb1 WT RNA-seq Rep2read2

description:
polyA+ RNA
RNA seq of polyA+ RNA random primed

data_processing:
RNA-Seq reads were aligned using TophHat v1.4.1 (Pair-ends were treated as single-ends)
GRO-Seq reads were aligned using Bowtie v0.12.7
Log2 differential expressions of RNA-Seq were calculated using edgeR v3.7.17
profiles of Gro-Seq were calculated using 50bp bin counting
Genome_build: GRCh37/hg19 assembly
Supplementary_files_format_and_content: GRO-Seq: bigWig
Supplementary_files_format_and_content: RASL-Seq and RNA-Seq: bigBed (log2 fold change at 7th column)


title: Col0 replicate 2 mRNA

source_name: Aerial tissue from 14 days old seedlings_Col0


title: Col0 replicate 1 mRNA


title: Rpb1 WT RNA-seq Rep1read2


title: fringe1/2 BS-seq

description: Bisulfite-treated

data_processing:
BS-Seq reads were mapped with BS seeker (Chen et al., 2010 BMC Bioinformatics) to TAIR10 chr1..5/C/M, allowing up to 2 mismatches and retaining only uniquely-mapping reads. Identical reads were collapsed into single reads. Only reference genome cytosines covered with at least 4 reads were considered for analysis.
Genome_build: TAIR10
Supplementary_files_format_and_content: Presented as a single base-pair resolution UCSC genome browser wiggle file for each nuclear genome (1-5), where column 1 is the genomic coordinate of the cytosine and column 2 is the methylation level #C/(#C+#T). For each genotype, three wiggle files were generated. Where each file represents data of methylation levels in three sequence contexts (CG, CHG, CHH).

source_name: 3wk-old leaf tissue

extract_protocol: Fragmented genomic DNA library as described in Stroud et al., Cell 2013; 152(1-2):352-64.

treatment_protocol: Genomic DNA was extracted using Qiagen DNeasy Mini Kit.

value: frg1 frg2

tag: ecotype

value: Columbia


title: Rpb1  R749H GRO-seq washout 20'


title: Rpb1 WT RNA-seq Rep2read1

value: doxycycline induced, a-amanitin (2.5 mg/ml) 42 hr


title: Rpb1 R749H RNA-seq Rep2read2

description:
polyA+ RNA
RNA seq of polyA+ RNA random primed
supplementary file: exons.WT_vs_C4.dj.gz
supplementary file: rintrons.WT_vs_C4.dj.gz


title: frg1 frg2 replicate 3 mRNA


title: Rpb1 R749H RNA-seq Rep2read1


title: Rpb1 R749H RNA-seq Rep1read2


title: Rpb1 E1126G RNA-seq Rep2read2

description:
polyA+ RNA
RNA seq of polyA+ RNA random primed
supplementary file: exons.WT_vs_E1126g.dj.gz
supplementary file: rintrons.WT_vs_E1126g.dj.gz


title: Rpb1 H1106Y GRO-seq control


title: Col0 replicate 3 mRNA


title: Rpb1 E1126G RNA-seq Rep1read1


title: Rpb1 E1126G RNA-seq Rep1read2


title: P. chlororaphis_scv_AB_rep2

city: Vernon Hills

first: DONGPING

last: WANG

state: IL

address: 777 Forest Edge Dr

department: Amlan

institute: OilDri Corporation of America

description:
strain: Pseudomonas chlororaphis 30-84 genotype: mutant, growing condition: AB minimal medium, Stage: late logarithm phase
Strand-specific cDNA libraries were constructed using the SOLiD Total RNA-Seq kit according to the manufacturer’s instructions with the following modifications: 1) For RNA fragmentation, 300 ng of rRNA-depleted RNA were incubated at 95˚C for 5 min. Bioanalyzer traces were used to confirm that the largest proportion of RNA fragments were around 200 bp. 2) Bioanalyzer traces following reverse transcription were used to confirm that hybrization and ligation of RNA adapters and subsequent reverse transcription of the RNA to cDNA was successful. 3) Amplification of cDNA was carried out following reverse transcription, but preceding the size-selection step. Samples were barcoded by replacing the SOLiD 3’ primer with the appropriate barcoding primer as directed in the SOLiD instructions. Size selection of DNA amplicons ranging from 200-300 bp was performed using an E-Gel iBase system (SYBR Safe 2% SizeSelect gel) (Life Technologies, Carlsbad, CA).  This step was repeated to ensure that adapter-adapter dimers were fully eliminated before sequencing. Paired-end sequencing was carried out by the University of Texas Genomic Sequencing and Analysis Facility (UTGSAF: http://www.ti3d.utexas.edu/facilities/gene-expression) on a Life Technologies SOLiD 5500xl sequencing system. Targeted sequencing depth was six-million paired-end reads per sample.

data_processing:
Paired-end sequencing was carried out by the Otogenetic company. Targeted sequencing depth was ten-million paired-end reads per sample.
Based on gene annotation information, the pipeline produced genome alignment results as a compressed binary version of the Sequence Alignment Map (BAM files).
Based on gene annotation information, the pipeline produced genome alignment results as a compressed binary version of the Sequence Alignment Map (BAM files).
Mapped reads were visualized using BamView in Artemis 13.2.0 (Rutherford et al. 2000).
The number of reads per kb of transcript per million mapped reads (RPKM) was used to normalize the raw data (Mortazavi et al., 2008), and mean RPKM values were determined for both the wild-type and mutant samples.
Genome_build: GenBank Accession #: PRJNA67533
Supplementary_files_format_and_content: Excel file containing the RPKM values for each gene

organism: Pseudomonas chlororaphis subsp. aureofaciens 30-84

source_name: SCV mutant cells

growth_protocol: Biological replicates of every strain were started from single colonies located on two separate plates containing AB + 2% CAA and then transferred to 1.5 ml AB + 2% CAA broth. All cultures were grown at 28˚C to an approximate OD600 = 1.8.

extract_protocol:
Transfer 700 μl lysate into RNeasy Mini column place in a 2 ml collection tube and centrifuge for 30 s at highest speed. Add 350 μl Buffer RW1 to the column and centrifuge for 30 s. Discard flow-through and reuse the collection tube. Add 10 μl DNase stock solution to 70 μ l RDD Buffer. Add the DNase I solution into the column and incubate at RT for 15 min. Add 350 μl Buffer RW1 to the column and wait for 5 min, centrifuge for 30 s. Discard the flow-through and collection tubes. Place the RNeasy Mini spin column in a new 2 ml collection tube. Add 500 μl Buffer RPE to the column. Centrifuge for 30 s. Place the column in a new 1.5 ml tube, and centrifuge for 1 min to eliminate any residue ethanol. Place the column in a new collection tube. Add 50 μl RNase free water, centrifuge for 1 min. Store RNA samples at -20°C
Strand-specific cDNA libraries were constructed using Illumina TruSeq RNA Sample Preparation Kits.

value: SCV mutant

tag: strain


title: P. chlororaphis_scv_AB_rep1


title: P. chlororaphis_WT_AB_rep2 (scv)

description:
strain: Pseudomonas chlororaphis 30-84 genotype: wild type, growing condition: AB minimal medium, Stage: late logarithm phase
Strand-specific cDNA libraries were constructed using the SOLiD Total RNA-Seq kit according to the manufacturer’s instructions with the following modifications: 1) For RNA fragmentation, 300 ng of rRNA-depleted RNA were incubated at 95˚C for 5 min. Bioanalyzer traces were used to confirm that the largest proportion of RNA fragments were around 200 bp. 2) Bioanalyzer traces following reverse transcription were used to confirm that hybrization and ligation of RNA adapters and subsequent reverse transcription of the RNA to cDNA was successful. 3) Amplification of cDNA was carried out following reverse transcription, but preceding the size-selection step. Samples were barcoded by replacing the SOLiD 3’ primer with the appropriate barcoding primer as directed in the SOLiD instructions. Size selection of DNA amplicons ranging from 200-300 bp was performed using an E-Gel iBase system (SYBR Safe 2% SizeSelect gel) (Life Technologies, Carlsbad, CA).  This step was repeated to ensure that adapter-adapter dimers were fully eliminated before sequencing. Paired-end sequencing was carried out by the University of Texas Genomic Sequencing and Analysis Facility (UTGSAF: http://www.ti3d.utexas.edu/facilities/gene-expression) on a Life Technologies SOLiD 5500xl sequencing system. Targeted sequencing depth was six-million paired-end reads per sample.

source_name: wild type bacterial cells

value: wild type


title: Rpb1 E1126G RNA-seq Rep2read1

source_name: HEK293 Flp-in T-REx pcDNA5 Rpb1 Amr E1126G


title: P. chlororaphis_WT_AB_rep1 (scv)


title: P. chlororaphis_WT_AB_rep2

data_processing:
Paired-end sequencing was carried out by the Otogenetic company. Targeted sequencing depth was ten-million paired-end reads per sample.
Based on gene annotation information, the pipeline produced genome alignment results as a compressed binary version of the Sequence Alignment Map (BAM files).
Mapped reads were visualized using BamView in Artemis 13.2.0 (Rutherford et al. 2000).
The number of reads per kb of transcript per million mapped reads (RPKM) was used to normalize the raw data (Mortazavi et al., 2008), and mean RPKM values were determined for both the wild-type and mutant samples.
Genome_build: ASM28191v1 (BioProject PRJNA67533)
Supplementary_files_format_and_content: Table containing the RPKM values for each gene

growth_protocol: Two biological replicates of every strain were started from single colonies located on two separate plates containing AB + 2% CAA and then transferred to 10 ml AB + 2% CAA broth. All cultures were grown at 28˚C with shaking (200 rpm) to an approximate OD600 = 2.0.

extract_protocol:
Transfer 700 μl lysate into RNeasy Mini column place in a 2 ml collection tube and centrifuge for 30 s at highest speed. Add 350 μl Buffer RW1 to the column and centrifuge for 30 s. Discard flow-through and reuse the collection tube. Add 10 μl DNase stock solution to 70 μ l RDD Buffer. Add the DNase I solution into the column and incubate at RT for 15 min. Add 350 μl Buffer RW1 to the column and wait for 5 min, centrifuge for 30 s. Discard the flow-through and collection tubes. Place the RNeasy Mini spin column in a new 2 ml collection tube. Add 500 μl Buffer RPE to the column. Centrifuge for 30 s. Place the column in a new 1.5 ml tube, and centrifuge for 1 min to eliminate any residue ethanol. Place the column in a new collection tube. Add 50 μl RNase free water, centrifuge for 1 min. Store RNA samples at -20°C
Strand-specific cDNA libraries were constructed using the SOLiD Total RNA-Seq kit according to the manufacturer’s instructions with the following modifications: 1) For RNA fragmentation, 300 ng of rRNA-depleted RNA were incubated at 95˚C for 5 min. Bioanalyzer traces were used to confirm that the largest proportion of RNA fragments were around 200 bp. 2) Bioanalyzer traces following reverse transcription were used to confirm that hybrization and ligation of RNA adapters and subsequent reverse transcription of the RNA to cDNA was successful. 3) Amplification of cDNA was carried out following reverse transcription, but preceding the size-selection step. Samples were barcoded by replacing the SOLiD 3’ primer with the appropriate barcoding primer as directed in the SOLiD instructions. Size selection of DNA amplicons ranging from 200-300 bp was performed using an E-Gel iBase system (SYBR Safe 2% SizeSelect gel) (Life Technologies, Carlsbad, CA).  This step was repeated to ensure that adapter-adapter dimers were fully eliminated before sequencing. Paired-end sequencing was carried out by the University of Texas Genomic Sequencing and Analysis Facility (UTGSAF: http://www.ti3d.utexas.edu/facilities/gene-expression) on a Life Technologies SOLiD 5500xl sequencing system. Targeted sequencing depth was six-million paired-end reads per sample.

treatment_protocol: wild type and mutant strains


title: P. chlororaphis_ZN_AB_rep1

source_name: Phenazine deficient mutant cells

value: ZN mutant


title: shRPA3 in ultramiR Replicate B

status: Public on Nov 24 2014

city: Cold Spring Harbor

first: Simon

middle: Robert Vincent

last: Knott

state: NY

address: 1 Bungtown Rd

department: Biology

institute: Cold Spring Harbor Laboratory

description:
size selected RNA
small RNAs from ultramiRharbored shRPA3 Infected cells
processed data file:  Rpa3_Variance_Stabalized.txt

data_processing:
1) Gallus gallus and Homo sapiens microRNA fasta files were obtained from miRbase. Illumina adapter sequences were then appended to each guide sequence and the resultant files compiled using bowtie-build.
2) Fastq files were allinged to the index files described above with bowtie. Allowing two mismatches.
3) shRNA and microRNA counts were dtermined using uniq -c.
4) Counts were normalized using the DESeq getVarianceStabalizedData function.
5) Normalized shRNA counts were normalized further by their log2 fold change vs. the top 33% most expressed microRNAs.
Genome_build: galGal4 and hg19
Supplementary_files_format_and_content: Rpa3_Variance_Stabalized.txt & Renilla_Variance_Stabalized.txt, Each file has 5 fields: 1) shRNA/microRNA, 2) mir30-shRNA infected shRNA/microRNA normalized counts (replicate A), 3) mir30-shRNA infected shRNA/microRNA normalized counts (replicate B),  4) ultramiR-shRNA infected shRNA/microRNA normalized counts (replicate A), 5) ultramiR-shRNA infected shRNA/microRNA normalized counts (replicate B)

organism: Gallus gallus

source_name: shRNA Infected ERC cells

growth_protocol:
ERC:DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin
HEK293T: DMEM high glucose supplemented with 10% fetal bovine serum, non-essential amino acids and penicillin streptomycin

extract_protocol:
RNA < 200 bps was isolated using the mirVana miRNA isolation kit (Life Technologies). F
Following RNA isolation, smallRNAs were cloned using the illumina sRNA kit.

treatment_protocol: Cells were infected at copy-number 1. Following infection cells were selected with G418 (1 mg/ml) until reaching 95% infection rate.

tag: rna type

value: small RNAs

value: ERC


title: shRPA3 in ultramiR Replicate A

description:
size selected RNA
small RNAs from ultramiR harbored shRPA3 Infected cells
processed data file:  Rpa3_Variance_Stabalized.txt


title: Rpb1 WT RNA-seq Rep1read1


title: shRPA3 in mir30 Replicate B

description:
size selected RNA
small RNAs from mir30 harbored shRPA3 Infected cells
processed data file:  Rpa3_Variance_Stabalized.txt


title: shRenilla in ultramiR Replicate B

description:
size selected RNA
small RNAs from ultramiRharbored shRenilla Infected cells
processed data file:  Renilla_Variance_Stabalized.txt

source_name: shRNA Infected HEK293  cells


title: shRenilla in ultramiR Replicate A

description:
size selected RNA
small RNAs from ultramiR harbored shRenilla Infected cells
processed data file:  Renilla_Variance_Stabalized.txt


title: shRenilla in mir30 Replicate B

description:
size selected RNA
small RNAs from mir30 harbored shRenilla Infected cells
processed data file:  Renilla_Variance_Stabalized.txt


title: shRenilla in mir30 Replicate A


title: P. chlororaphis_ZN_AB_rep2


title: Rpb1 R749H RNA-seq Rep1read1

source_name: HEK293 Flp-in T-REx pcDNA5 Rpb1 Amr R749H


title: Chromatin_associated

city: Copenhagen

first: Nicolai

last: Krogh

address: Blegdamsvej 3B

department: Department of Cellular and Molecular Medicine

country: Denmark

institute: University of Copenhagen

description: RNA was purified according to: F. Carrillo Oesterreich, S. Preibisch, K. M. Neugebauer, Global analysis of nascent RNA reveals transcriptional pausing in terminal exons. Mol Cell 40, 571 (2010).

data_processing:
Reads were identified by a 4 nucleotide barcode which was removed
Cutadapt v1.2 was used to remove the 3’ adaptor and discard reads shorter than 15 nucleotides
The trimmed reads were mapped using the Bowtie 2 aligner v 2.1.0 with default settings to the precursor ribosomal RNA (35S rRNA) gene of Saccharomyces cerevisiae s288c (RDN37-1;SGD:S000006486, GeneID: 9164931)
Only reads mapping uniquely and with a minimum of three correct nucleotides at the 5’ or 3’ end were used to compute the number of 3’ read ends (corresponding to the 5’ nt in the sequence insert) and 5’ read ends (corresponding to the 3’ nt in the sequence insert), respectively. In the latter, the counts were shifted one position upstream at the level of the sequence read in order to get the 3’ and 5’ read ends to align to the methylated nucleotide in the reference sequence.
Various scoring systems were used to detect and visualise 2'-O-methylation based on the 5' and 3' read end counts.
Genome_build: n/a, Saccharomyces cerevisiae s288c (RDN37-1;SGD:S000006486, GeneID: 9164931)
Supplementary_files_format_and_content: The processed data files are bed files for visualising the data in the UCSC browser. Each file contains 6 tracks: 5' RNA ends, 3' RNA ends, 5' and 3'. RNA ends combined, score A, score B and Pseudo-U score as described in Birkedal et al. The bed-files may be uploaded to the UCSC browser individually or in combination. The file generic_ribo_info.bed should be included at all times to navigate known RNA modifications.

organism: Saccharomyces cerevisiae

source_name: Yeast

extract_protocol:
RNA was extracted using hot phenol protocol as described in Birkedal et al. Chromatin associated RNA was purified according to: F. Carrillo Oesterreich, S. Preibisch, K. M. Neugebauer, Global analysis of nascent RNA reveals transcriptional
The fragments are ligated to RNA oligos using a tRNA ligase that has been mutated to remove its kinase activity, reverse transcribed and the cDNA used as input for sequencing.

tag: modification

value: Chromatin associated RNA


title: shRPA3 in mir30 Replicate A


title: P. chlororaphis_WT_AB_rep1


title: shRNA Slpi2 Replicate B_1

description: processed data file: Slpi.variance_stabalized

data_processing:
1) Reads were alligned to the mm10 genome using bowtie-2.
2) Each gene's read counts were determined using htseq -count (python) function.
3) Reads were normalized across samples using DESeq (bioconductor)
mgp_variance_stabalized.txt and mgp_stabalized.txt each have 11 fields.
mgp_variance_stabalized
1) Gene Name
2) Normalized Counts for cells infected with 88943 (replicate A)
3) Normalized Counts for cells infected with 88943 (replicate B)
4) Normalized Counts for cells infected with Mgp1 (replicate A)
5) Normalized Counts for cells infected with Mgp1 (replicate B)
6) Normalized Counts for cells infected with Mgp1U1 (replicate A)
7) Normalized Counts for cells infected with Mgp1U1 (replicate B)
8) Normalized Counts for cells infected with Mgp1U3 (replicate A)
9) Normalized Counts for cells infected with Mgp1U3 (replicate B)
10) Normalized Counts for cells infected with Mgp2 (replicate A)
11) Normalized Counts for cells infected with Mgp2 (replicate B)
slpi_variance_stabalized
1) Gene Name
2) Normalized Counts for cells infected with 66708 (replicate A)
3) Normalized Counts for cells infected with 66708 (replicate B)
4) Normalized Counts for cells infected with Slpi1U1 (replicate A)
5) Normalized Counts for cells infected with Slpi1U1 (replicate B)
6) Normalized Counts for cells infected with Slpi1U2 (replicate A)
7) Normalized Counts for cells infected with Slpi1U2 (replicate B)
8) Normalized Counts for cells infected with Slpi2 (replicate A)
9) Normalized Counts for cells infected with Slpi2 (replicate B)
10) Normalized Counts for cells infected with Slpi4  (replicate A)
11) Normalized Counts for cells infected with Slpi4  (replicate B)
Genome_build: mm10
Supplementary_files_format_and_content: Normalized counts for cells infected with indicated shRNA.

organism: Mus musculus

source_name: Mouse 4T1 Cell Line

growth_protocol: DMEM high glucose (Life Technologies) supplemented with 5% fetal bovine serum (Thermo Scientific), 5% fetal calf serum (Thermo Scientific), non-essential amino acids (Life Technologies) and penicillin streptomycin (Life Technologies)

extract_protocol:
RNA was purified and DNAse treated using the Qiagen RNeasy Mini Kit
The NuGEN Ovation RNA-Seq V2 protocol was carried out on 100 ng of total RNA. cDNA was fragmented using the Covaris LE220 sonicator according to the manufacturer’s instruction to yield a target fragment size of 200 bp. The fragmented cDNA was subsequently processed using the NuGEN Ovation Ultralow DR Multiplex System. Libraries were analyzed on an Illumina Hiseq 2 using 76bp reads.

treatment_protocol: Cells were infected at 0,3 MOI and selected to >95% infection percentage (averge of 10 days). shRNA-66708 abd 88943 infected cells were selexted with 1 μg/ml Puromycin . All other shRNA infected cells were selected with 500 μg/ml hygromycin.

tag: cell treatment

value: shRNA Infected Cells


title: shRNA Slpi4 Replicate A_1


title: shRNA Slpi4 Replicate B_2


title: shRNA Slpi2 Replicate B_2


title: shRNA Slpi4 Replicate A_2


title: shRNA Slpi4 Replicate B_1


title: shRNA Slpi2 Replicate A_1


title: shRNA Slpi1U2 Replicate B_1


title: shRNA Slpi1U2 Replicate A_1


title: shRNA Slpi2 Replicate A_2


title: shRNA Slpi1U2 Replicate B_2


title: shRNA Slpi1U2 Replicate A_2


title: shRNA Slpi1U1 Replicate B_1


title: shRNA Slpi1U1 Replicate B_2


title: P. chlororaphis_WT_AB_rep3 (scv)


title: shRNA Mgp2 Replicate B_1

description: processed data file: Mgp.variance_stabalized


title: shRNA Slpi1U1 Replicate A_1


title: shRNA Mgp2 Replicate B_2


title: shRNA Mgp2 Replicate A_2


title: shRNA Mgp1U3 Replicate B_2


title: shRNA Mgp1U3 Replicate A_2


title: shRNA Mgp1U3 Replicate B_1


title: shRNA Mgp1U1 Replicate B_2


title: shRNA Mgp1U3 Replicate A_1


title: shRNA Mgp1U1 Replicate A_2


title: shRNA Mgp1U1 Replicate B_1


title: shRNA 88943 Replicate B_2


title: shRNA Mgp1 Replicate A_2


title: shRNA Mgp1 Replicate B_2


title: shRNA Mgp1U1 Replicate A_1


title: shRNA Mgp1 Replicate B_1


title: shRNA Slpi1U1 Replicate A_2


title: shRNA 88943 Replicate B_1


title: shRNA 66708 Replicate B_1


title: shRNA 66708 Replicate A_2


title: shRNA 88943 Replicate A_1


title: shRNA 88943 Replicate A_2


title: L282_H06

city: Stockholm

first: Sten

last: Linnarsson

address: Scheeles väg 1

department: Medical Biochemistry and Biophysics

country: Sweden

institute: Karolinska Institutet

description: single cell

data_processing:
Read quality filtering and barcode extraction: Every read marked as valid by the Illumina control software was processed as follows: a) any 3' bases with a quality score of 'B' were removed; b) bases 1-10 at the 5', consisting of 4 bases UMI followed by 6 bases well-identifying barcode were extracted from the read. If any of the four UMI bases had a Phred score < 17 the read was discarded; c) the following bases should be template switch derived Gs. If the first three were not all Gs or two Gs and one uncalled base, the read was discarded. Else, these plus a maximum of six more consecutive Gs were removed; d) the remaining sequence should be transcript derived. If this ended in a poly(A)-sequence leaving less than 25 bases, the read was discarded; e) if the transcript-derived sequence consisted of less than six non-A bases, or a dinucleotide repeat with less than six other bases at either end, the read was discarded.
Alignment:the reads were aligned to the genome using the Bowtie1 aligner, allowing for up to three mismatches and up to 24 alternative mappings for each read. The genome included an artifical chromosome, containing a concatamer of the ERCC control sequences. Any reads with no alignments were re-aligned against another artifical chromosome, containing all possible splice junctions arising from the exons defined by the known transcript variants. Reads mapping within these splice junctions were translated back to the corresponding actual genomic positions. The UCSC transcript models were used for the expression level calculation. If a locus had several transcript variants, the exons of these were merged to a combined model that represented all expression from the locus. To account for incompete cap site knowledge, the 5' ends of all models were extended by 100 bases, but not beyond the 3' end of any upstream nearby exon of another gene of the same orientation.
Annotation and quantitation: The annotation step was performed barcode-by-barcode. Only reads mapping in sense orientation of an exon were considered as potentially transcript derived. Any such read that had one or more repeat mappings that was outside exons, was assigned randomly as one of these repeats and contributed to the summarized read count of that repeat class. Else, if it had one or more sense mappings to exons, it was assigned at the exon where it was closest to the transcript model 5' end, even if the sequence was repeat-like. If it had no exon mapping, it was assigned randomly at one of the mappings. The expression level of each transcript model was taken as the total number of reads in sense orientation at all its possible mapping positions.
Genome_build: UCSC mm9
Supplementary_files_format_and_content: Usoskin_DataTable.txt is a TAB-delimted text file containing Reads Per Million values for each analyzed feature in each well. Prefix 'r_' indicates repeat types and summarizes total expression from all repeat regions (from RepeatMasker) of that type. Suffix '_locX', X=1,2... indicates alternative loci expressing the same gene.

source_name: lumbar dorsal root ganglion

growth_protocol: 6-8 week old C57/Bl6 mice

extract_protocol:
Six lumbar DRGs (L4-L6) were dissected and dissociated using papain and collagenase/dispase followed by Percoll gradient centrifugation. The sample was always kept on ice apart from enzyme incubation. In some preparations a cooled microscope stage was used. The final cell pellet was suspended in 300 ml of L15 medium supplemented with B27. Cells were picked on an in house constructed picking setup built around an inverted Nikon microscope. Individual cells were sucked into a glass capillary attached to a CellTram syringe with visual control that individual cells were captured. The capillary tip was positioned in a well of a 96 well plate containing 3 microlitre of lysis buffer into which the cell was released by operating the CellTram syringe. 48 random cells were picked at each time within 40-60 minutes. The lysis buffer contained 20mMTris-HCl at pH8.0, 75 mM KCl, 6 mM MgCl2, 0.02% Tween-20 with 400 nM STRT-V3-T30 (5'-biotin-AAGCAGTGGTATCAACGCAGAGTCGACT(30)VN-3') and 400 nM STRT-V2-n (5'-AAGCAGTGGTATCAACGCAGAGTGCAGTGCTXXXXXXrGrGrG-3', where ‘‘rG’’ denotes a riboguanine and ‘‘XXXXXX’’ was a 6-bp barcode). Each well of the capture plate contained a different template-switching helper oligo with a distinct barcode. Plates were then frozen.
The cell capture plate was thawed, and 5 microlitre reverse transcription mix (4mM DTT, 2mM dNTP, 5U/microlitre SuperscriptII in 100 mM Tris-HCl at pH 8, 375 mM KCl, 0.1% Tween-20, 6 mM MnCl2, 2500 molecules of control mRNA) was added to each well. The synthetic mRNA consisted of 92 different ERCC control RNAs). The plate was incubated (10°C for 10 min, 42°C for 45 min) to complete reverse transcription and template switching. To purify the cDNA and remove unreacted primers, 100 microlitre MyOne carboxylatebeads (Invitrogen) was washed twice in 100 microlitre EBT (10 mM Tris-Cl at pH 8.5, 0.02% Tween-20) and then resuspended in 2 mL 14% PEG-6000 in 0.9 M NaCl, and 20 microlitre of this mixture was added to each well. Beads from all wells were pooled, washed in 70% ethanol twice, dried, and eluted in 37 microlitre EBT in a 1.5-mL polyallomer tube (Beckman). The cDNA was amplified in a single tube in 50 microlitre of 200 microM dNTP, 200 nM STRT-PCR primer (5’-biotin-AAGCAGTGGTATCAACGCAGAGT-3’), 1x Advantage2 DNA Polymerase Mix (Clontech) in 1x Advantage2 PCR buffer (Clontech) with 1 min at 95°C followed by 20 cycles of 15 sec at 95°C, 30 sec at 65°C, 4 min at 68°C,with heated lid.A 5 microlitre aliquot was amplified for another five cycles and visualized on a 1.2% agarose E-gel (Invitrogen) to confirm the range of cDNA lengths. The product was immobilized on MyOne C1 Streptavidin beads. Twenty microliters of beads was washed twice in 50 microlitre 2x BWT (10 mM Tris HCl at pH 7.5, 1 mM EDTA, 2 M NaCl, 0.02% Tween-20) and added to the remaining 45 microlitre PCR product. After a 10-min incubation at room temperature, the beads were washed three times in 50 microlitre 1x BWT and twice in EBT. Amplified cDNA was fragmented by DNase I in the presence of Mn2+. Beads were resuspended in DNase I buffer supplemented with 10 mM MnCl2 and DNaseI diluted to 0.0003U/microlitre in a total volume of 120 microlitre for exactly 8 min at room temperature. The reaction was stopped by washing the beads five times in 50 microlitre EBT. Beads were resuspended in 25 microlitre EBT, and 25 microlitre NEBNext End Repair reaction mix (New England Biolabs) was added; then the beads were incubated for 30 min at room temperature. The beads were washed twice in EBT, then resuspended in 21 microlitre EBT; 2.5 microlitre NEBNext dA tailing buffer and 1.5 microlitre Klenow exo- (both NEB) were added, and the reaction was incubated for 30 min at 37°C, followed by two washes in 50 microlitre EBT. An adapter containing the Illumina P2 sequence (5’-CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT-3’ and 3’-PHO-GTTCGTCTTCTGCCGTATGCTCGAGAAGGCTAG-PHO-5’) was ligated by resuspending the beads in 25 microlitre EBT and adding 2x ligationmix (2x NEBuffer4, 2 microM adapter,1U/microlitre T4DNAligase,2U/microlitre SalI-HF, and 2mM ATP; all reagents from NEB) and incubating for 30min at 37°C and then washing twice in 25 microlitre EBT. The beads were then used as template in a 100 microlitre PCR reaction (200 microM dNTP, 400 nM each primer of primers 5’-AATGATACGGCGACCACCGAGATCTAAGCAGTGGTATCAACGCAGAGT-3’ and 5’-CAAGCAGAAGACGGCATACGAG-3’, 200 microM dNTP ,0.2U/microlitre Phusion polymerase in PhusionHF buffer ;all from NEB) with 30 sec at 98°C, followed by 12 cycles of [10 sec at 98°C, 30 sec at 65°C, 30 sec at 72°C] followed by 5 min at 72°C. The product was purified by AmPure XP (Beckman Coulter) and resuspended in 20 microlitre EBT. The sample was size-selected on a 2% E-gel with SYBR Safe (Invitrogen), recovering the 200 400 bp range by Qiaquick Gel Extraction (replacing the heating step with 15 min of vigorous agitation). The well indexing barcodes are read as bases 1-6 of the read, or for plates L281 and L282 as bases 5-10 of the read.

tag: cell picking session

value: C

tag: cell picking temperature

value: Cooling

tag: gender

value: male


title: shRNA 66708 Replicate A_1


title: shRNA Mgp2 Replicate A_1


title: L282_H03

description: single cell


title: L282_H04

description: medium


title: L282_H02

description: single cell


title: L282_G05

description: single cell


title: L282_G03

description: single cell


title: L282_G01

description: single cell


title: L282_G02

description: single cell


title: L282_F06

description: medium


title: L282_F03

description: single cell


title: shRNA 66708 Replicate B_2


title: L282_F04

description: single cell


title: L282_F05

description: >1 cell


title: L282_F01

description: >1 cell


title: L282_E05

description: >1 cell


title: L282_F02

description: medium


title: L282_E04

description: single cell


title: L282_H01

description: single cell


title: L282_H05

description: single cell


title: L282_E01

description: >1 cell


title: L282_D06

description: medium


title: L282_E02

description: medium


title: L282_D03

description: medium


title: L282_E03

description: single cell


title: L282_C06

description: single cell


title: L282_D05

description: single cell


title: L282_G04

description: single cell


title: L282_G06

description: single cell


title: L282_D04

description: single cell


title: L282_C03

description: single cell


title: L282_D01

description: single cell


title: L282_C04

description: single cell


title: L282_E06

description: single cell


title: L282_C05

description: single cell


title: L282_C02

description: single cell


title: L282_C01

description: single cell


title: L282_B06

description: single cell


title: L282_B04

description: single cell


title: L282_B05

description: single cell


title: L282_B03

description: single cell


title: L282_A03

description: medium


title: L282_B01

description: single cell


title: L282_A05

description: single cell


title: L282_A06

description: single cell


title: L282_A01

description: single cell


title: L281_H05

description: >1 cell


title: L281_H06

description: empty well


title: L282_A02

description: single cell


title: L282_B02

description: single cell


title: L281_H04

description: >1 cell


title: shRNA Mgp1 Replicate A_1


title: L281_G06

description: medium


title: L281_H01

description: single cell


title: L282_A04

description: single cell


title: L281_H02

description: single cell


title: L281_G04

description: >1 cell


title: L281_G03

description: single cell


title: L281_G02

description: single cell


title: L281_F06

description: single cell


title: L281_G01

description: single cell


title: L281_F05

description: single cell


title: L282_D02

description: single cell


title: L281_F04

description: single cell


title: L281_F03

description: single cell


title: L281_F02

description: >1 cell


title: L281_H03

description: single cell


title: iRaf ES

first: Felix

last: Krueger

address: Granta Park

department: Bioinformatics

institute: Altos Labs

data_processing:
Genomic mapping of paired-end reads was performed with Bowtie (v0.12.8) using the following parameters: -m 1 --strata --best -X 700. Reads were mapped to the mouse genome build NCBIM37. Final data analysis was performed using SeqMonk software (www.bioinformatics.babraham.ac.uk).
Genome_build: NCBIM37
genome-wide quantification file: Quantitated MeDIP-Seq files were generated for 1kb non-overlapping genomic tiles using SeqMonk (v0.28.0). The result file carries descriptive column headers and is tab-delimited; the relevant columns are: (1) ID; (2) Chromosome; (3) Start; (4) End; (13-21) Raw read count per 1kb tile

source_name: Embryonic Stem Cells

growth_protocol: Prior to the start of the reprogramming protocol, ES and TS cells were cultured in routine conditions on a layer of irradiated mouse embryonicfibroblasts (MEFs). ES cells were maintained in serum+LIF conditions, and TS cells in 70% MEF-conditioned media, 30% TS media, with 25ng/ml bFGF and 1µg/ml Heparin.

extract_protocol:
At the end of the trans-differentiation time-course, cells were FACS-purified to remove MEFs, and DNA extracted with phenol-chloroform according to standard protocols. DNA was used for MeDIP analysis as described previously (Senner et al., Stem Cells 2012).
Genomic DNA was fragmented by sonication (Diagenode Bioruptor), and adaptor ligated with paired-end adaptors 5’-PGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG-3’ and 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3’ (* Phosphorothioate group) and the NEBnext library preparation kit. Subsequently immunoprecipitaions with an anti-5-methylcytosine antibody were carried out in triplicate. IP triplicates –prepared for each cell type – were pooled and MeDIP DNA libraries were column-purified (MinElute, Qiagen) and eluted in 17μl of EB buffer (pre-warmed at 55ºC). DNA unbound fractions were processed in parallel. MeDIP libraries (and unbound DNA fractions, as control) were amplified by PCR for 12 cycles using distinct combinations of primers: PE 1.0 (Fw) AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT; PE 2.0 (Rv) CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT; iPCR tag6 (Rv) CAAGCAGAAGACGGCATACGAGATACATTGGCGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATC; iPCR tag7 (Rv) CAAGCAGAAGACGGCATACGAGATCAGATCTGGAGA  TCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATC; iPCR tag8 (Rv) CAAGCAGAAGACGGCATACGAGATCATCAAGTGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATC. Amplified MeDIP libraries (and unbound DNA fractions, as control) were  resolved by 2% agarose gel electrophoresis, and DNA fragments within the 300-500 nt range were cut out and DNA was purified using the Qiagen Gel extraction kit. 5mC immuno-precipitation efficiency was confirmed by analysing ES and TS  cell MeDIP pre- and post-amplification libraries by quantitative PCR for candidate loci (Elf5, Nanog). Library concentration was determined using KAPA Illumina SYBR Universal Lid Q Kit (ANACHEM) and by Bioanalyzer.

treatment_protocol: During the experimental time course of trans-differentiation, cells were plated on MEFs in TS cell conditions as specified with the appropriate inducer (Dox or 4HT) at a seeding density of approximately 1x10E4 cells/25cm2. Media was changed every 2 days. Cells were trypsinized after 6 days of culture and re-plated, again at 1x10E4 cells/25cm2 in TS conditions, for another 6 days.

tag: cell type

value: Embryonic Stem Cells

value: E14 - iRaf

tag: condition

value: 12 days of reprogramming in TS conditions


title: L281_F01

description: single cell


title: control ES

value: E14 - EGFP vector only


title: iRas ES

value: E14 - iRas


title: L281_G05

description: single cell


title: ES_E14_on_MEFs

value: Conventional, grown on MEFs


title: TS-Rs26

source_name: Trophoblast Stem Cells

value: Trophoblast Stem Cells


title: L281_E06

description: >1 cell


title: L281_E05

description: >1 cell


title: L281_E04

description: single cell


title: L281_E03

description: >1 cell


title: L142_H08

description: single cell

data_processing:
Read quality filtering and barcode extraction: Every read marked as valid by the Illumina control software was processed as follows: a) any 3' bases with a quality score of 'B' were removed; b) the 6 base well-identifying barcode was extracted from the 5' end of the read; c) the following bases should be template switch derived Gs. If the first three were not all Gs or two Gs and one uncalled base, the read was discarded. Else, these plus a maximum of six more consecutive Gs were removed; d) the remaining sequence should be transcript derived. If this ended in a poly(A)-sequence leaving less than 25 bases, the read was discarded; e) if the transcript-derived sequence consisted of less than six non-A bases, or a dinucleotide repeat with less than six other bases at either end, the read was discarded.
Alignment:the reads were aligned to the genome using the Bowtie1 aligner, allowing for up to three mismatches and up to 24 alternative mappings for each read. The genome included an artifical chromosome, containing a concatamer of the ERCC control sequences. Any reads with no alignments were re-aligned against another artifical chromosome, containing all possible splice junctions arising from the exons defined by the known transcript variants. Reads mapping within these splice junctions were translated back to the corresponding actual genomic positions. The UCSC transcript models were used for the expression level calculation. If a locus had several transcript variants, the exons of these were merged to a combined model that represented all expression from the locus. To account for incompete cap site knowledge, the 5' ends of all models were extended by 100 bases, but not beyond the 3' end of any upstream nearby exon of another gene of the same orientation.
Annotation and quantitation: The annotation step was performed barcode-by-barcode. Only reads mapping in sense orientation of an exon were considered as potentially transcript derived. Any such read that had one or more repeat mappings that was outside exons, was assigned randomly as one of these repeats and contributed to the summarized read count of that repeat class. Else, if it had one or more sense mappings to exons, it was assigned at the exon where it was closest to the transcript model 5' end, even if the sequence was repeat-like. If it had no exon mapping, it was assigned randomly at one of the mappings. The expression level of each transcript model was taken as the total number of reads in sense orientation at all its possible mapping positions.
Genome_build: UCSC mm9
Supplementary_files_format_and_content: Usoskin_DataTable.txt is a TAB-delimted text file containing Reads Per Million values for each analyzed feature in each well. Prefix 'r_' indicates repeat types and summarizes total expression from all repeat regions (from RepeatMasker) of that type. Suffix '_locX', X=1,2... indicates alternative loci expressing the same gene.

value: B

value: RT


title: L142_H07

description: single cell


title: L142_H09

description: single cell


title: L142_H11

description: single cell


title: L142_H10

description: single cell


title: L281_E01

description: >1 cell


title: Oct4-cKO


title: L142_H06

description: single cell


title: L142_H05

description: single cell


title: L142_H03

description: single cell


title: L281_E02

description: single cell


title: L142_H02

description: single cell


title: L142_H01

description: single cell


title: L281_D06

description: single cell


title: L142_G12

description: single cell


title: L142_G11

description: single cell


title: L142_G10

description: medium


title: L281_D05

description: empty well


title: L142_G08

description: single cell


title: L142_H12

description: single cell


title: L281_D04

description: >1 cell


title: L142_G07

description: single cell


title: L142_G05

description: single cell


title: L142_G09

description: single cell


title: L281_D03

description: >1 cell


title: L142_G06

description: single cell


title: L281_D02

description: >1 cell


title: L142_H04

description: single cell


title: L142_G04

description: single cell


title: L142_G02

description: single cell


title: L142_G01

description: single cell


title: L142_G03

description: single cell


title: L142_F11

description: single cell


title: L142_F10

description: single cell


title: L281_C06

description: single cell


title: L142_F12

description: single cell


title: L281_D01

description: >1 cell


title: L281_C05

description: single cell


title: L142_F08

description: single cell


title: L142_F09

description: single cell


title: L281_C04

description: >1 cell


title: L281_C03

description: >1 cell


title: L142_F06

description: single cell


title: L281_C01

description: single cell


title: L281_B06

description: >1 cell


title: L142_F05

description: single cell


title: L142_F04

description: single cell


title: L142_F03

description: single cell


title: L142_F02

description: single cell


title: L142_E11

description: single cell


title: L142_E12

description: single cell


title: L281_B05

description: >1 cell


title: L142_E10

description: single cell


title: L281_B04

description: single cell


title: L142_E09

description: single cell


title: L281_B03

description: single cell


title: L142_E08

description: single cell


title: L281_B02

description: single cell


title: L142_F07

description: single cell


title: L142_E03

description: single cell


title: L142_E04

description: single cell


title: L142_E05

description: single cell


title: L142_E06

description: single cell


title: L142_E02

description: single cell


title: L281_C02

description: >1 cell


title: L281_B01

description: >1 cell


title: L142_E07

description: single cell


title: L281_A06

description: single cell


title: L142_E01

description: single cell


title: L142_F01

description: single cell


title: L142_D11

description: single cell


title: L142_D12

description: single cell


title: L281_A05

description: single cell


title: L142_D10

description: single cell


title: L142_D09

description: single cell


title: L142_D07

description: single cell


title: L281_A03

description: >1 cell


title: L281_A04

description: >1 cell


title: L142_D06

description: single cell


title: L281_A02

description: >1 cell


title: L142_D08

description: single cell


title: L142_D05

description: single cell


title: L142_D03

description: single cell


title: L281_A01

description: >1 cell


title: L142_D01

description: single cell


title: L142_D02

description: single cell


title: Rpb1 WT RASL-seq Rep3

description:
polyA+ RNA
RASL- seq of polyA+ RNA

source_name: HEK293 Flp-in T-REx pcDNA5 Rpb1 Amr WT

value: doxycycline induced, a-amanitin (2.5 mg/ml) 42 hr


title: L142_C11

description: single cell


title: L142_C12

description: single cell


title: L228_H11

description: single cell


title: L228_H10

description: single cell


title: L228_H12

description: empty well


title: L142_C06

description: single cell


title: L142_C10

description: single cell


title: L142_C09

description: single cell


title: L142_C08

description: single cell


title: L228_H08

description: single cell


title: L142_C05

description: single cell


title: L142_C03

description: single cell


title: L228_H09

description: single cell


title: L142_C04

description: single cell


title: L228_H07

description: single cell


title: L142_D04

description: single cell


title: L228_H06

description: empty well


title: L142_C01

description: single cell


title: L142_C02

description: single cell


title: L228_H05

description: single cell


title: L228_H04

description: single cell


title: L142_B11

description: single cell


title: L142_B12

description: single cell


title: L142_C07

description: single cell


title: L142_B09

description: single cell


title: L142_B10

description: single cell


title: L142_B07

description: single cell


title: L228_H03

description: single cell


title: L142_B08

description: single cell


title: L228_H01

description: single cell


title: L142_B03

description: single cell


title: L142_B02

description: single cell


title: L142_B05

description: single cell


title: L142_B04

description: single cell


title: L142_A12

description: single cell


title: L228_H02

description: single cell


title: L228_G11

description: single cell


title: L142_B06

description: single cell


title: L228_G12

description: single cell


title: L142_B01

description: medium


title: L142_A10

description: single cell


title: L228_G10

description: single cell


title: L142_A09

description: single cell


title: L142_A07

description: >1 cell


title: L142_A08

description: single cell


title: L142_A06

description: single cell


title: L142_A05

description: single cell


title: L228_G08

description: single cell


title: L228_G09

description: single cell


title: L142_A04

description: single cell


title: L142_A03

description: single cell


title: L142_A02

description: single cell


title: L142_A01

description: empty well


title: L228_G07

description: single cell


title: L142_A11

description: single cell


title: L228_G06

description: empty well


title: L228_G05

description: single cell


title: L141_H11

description: single cell


title: L141_H10

description: single cell


title: L228_G04

description: single cell


title: L228_G03

description: single cell


title: L141_H09

description: single cell


title: L228_G02

description: single cell


title: L141_H07

description: single cell


title: L141_H08

description: single cell


title: L141_H06

description: single cell


title: L228_G01

description: single cell


title: L141_H05

description: single cell


title: L141_H04

description: single cell


title: L141_H03

description: single cell


title: L228_F12

description: single cell


title: L228_F11

description: single cell


title: L141_G10

description: single cell


title: L141_G12

description: medium


title: L141_G11

description: single cell


title: L228_F09

description: single cell


title: L141_H12

description: empty well


title: L141_G07

description: single cell


title: L228_F10

description: single cell


title: L141_G06

description: single cell


title: L141_G04

description: single cell


title: L228_F08

description: single cell


title: L141_G08

description: single cell


title: L141_G03

description: single cell


title: L141_H02

description: single cell


title: L228_F07

description: single cell


title: L141_G05

description: single cell


title: L141_G02

description: single cell


title: L228_F06

description: single cell


title: L141_H01

description: single cell


title: L141_F10

description: single cell


title: L141_F12

description: single cell


title: L141_F09

description: single cell


title: L141_G09

description: single cell


title: L141_F11

description: single cell


title: L141_F07

description: single cell


title: L228_F04

description: single cell


title: L141_F08

description: single cell


title: L141_F06

description: single cell


title: L228_F03

description: single cell


title: L141_F05

description: single cell


title: L228_F01

description: single cell


title: L141_F04

description: single cell


title: L141_F03

description: single cell


title: L141_F02

description: single cell


title: L141_F01

description: single cell


title: L141_G01

description: single cell


title: L141_E12

description: single cell


title: L228_F05

description: single cell


title: L228_E12

description: single cell


title: L141_E11

description: single cell


title: L228_E11

description: single cell


title: L141_E08

description: single cell


title: L141_E10

description: single cell


title: L141_E09

description: single cell


title: L228_E09

description: single cell


title: L141_E06

description: single cell


title: L141_E07

description: single cell


title: L141_E05

description: single cell


title: L228_E08

description: single cell


title: L141_E03

description: single cell


title: L228_E10

description: single cell


title: L141_E02

description: single cell


title: L141_D12

description: single cell


title: L228_F02

description: single cell


title: L141_E04

description: single cell


title: L141_E01

description: single cell


title: L141_D11

description: single cell


title: L228_E05

description: single cell


title: L141_D10

description: single cell


title: L141_D09

description: single cell


title: L141_D08

description: single cell


title: L141_D07

description: single cell


title: L228_E03

description: single cell


title: L228_E02

description: single cell


title: L228_D12

description: single cell


title: L228_E01

description: single cell


title: L141_D05

description: single cell


title: L228_E06

description: single cell


title: L141_D06

description: single cell


title: L228_D11

description: single cell


title: L228_E07

description: single cell


title: L141_D04

description: single cell


title: L141_D01

description: single cell


title: L141_C12

description: single cell


title: L141_C11

description: single cell


title: L228_D10

description: single cell


title: L141_C10

description: single cell


title: L228_D09

description: single cell


title: L141_C08

description: single cell


title: L141_C09

description: single cell


title: L141_C07

description: single cell


title: L141_C06

description: single cell


title: L141_C05

description: single cell


title: L228_D08

description: single cell


title: L141_C03

description: single cell


title: L141_C02

description: single cell


title: L141_D03

description: single cell


title: L228_E04

description: single cell


title: L141_D02

description: single cell


title: L228_D06

description: single cell


title: L141_C01

description: single cell


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title: ChIP-seq sample for siRNA knock-down of Tcf7l2: siRNA - Scrambled, 15 h, target protein - Hnf4a

city: Kuopio

first: Sami

last: Heikkinen

address: Yliopistonranta 1E

department: Institute of Biomedicine

country: Finland

institute: University of Eastern Finland

data_processing:
RNA-seq, transcriptome building: Pooled reads were mapped, using Tophat version 2.0.4 (with underlying Bowtie version 0.12.8), against UCSC known transcripts for rn4, supplemented with additional transcripts from Ensembl, and allowing for the identification of novel exon-exon junctions for the reference genome (rn4 supplemented with EST-based sequence at chr1:262,210,923-262,210,894 to partial fill a gap including a part of an exon of Tcf7l2; corresponding fix was introduced also for the UCSC reference transcriptome). Essential Tophat command arguments were: --library-type fr-unstranded --prefilter-multihits --no-coverage-search -G <UCSC+Ensembl known transcripts>.
RNA-seq, transcriptome building: Transcript mappings were further processed using Cufflinks package version 2.0.2 with essential command arguments, for Cufflinks: --frag-bias-correct --multi-read-correct --upper-quartile-norm -g <UCSC+Ensembl known transcripts> --library-type fr-unstranded, and for Cuffcompare: -r <UCSC+Ensembl known transcripts> -R -d 20 -V <.gff output of Cufflinks>.
RNA-seq, transcriptome building: The initial transcriptome was cleaned by removing all transcripts that were identified as fully silent (FPKM=0), strandless (in Cuffcompare class codes u, o, x, and, if also single-exon, i), or likely artifacts (Cuffcompare class codes e, p, r, s and c). The transcripts that did not represent any known rn4 gene were analyzed against mouse (mm10), human (hg19), as well as an updated rat reference transcriptome sequences using discontiguous megablast with stringent cutoffs to provide official gene symbols for a few additional genes.
RNA-seq, timecourse: Reads for time points 3, 6, 9, 12, 15, 18, 48, and 96 hours after Tcf7l2 or scramble siRNA treatment were mapped against the H4IIE-specific transcriptome using Tophat version 2.0.4 (with underlying Bowtie version 0.12.8). Essential Tophat command arguments were: --library-type fr-unstranded --prefilter-multihits --no-novel-juncs --transcriptome-only -G <H4IIE-specific transcriptome>.
RNA-seq, timecourse: Conversion to sorted BAM format using samtools
RNA-seq, timecourse: Differential expression was analyzed using Cuffdiff version 2.0.2., comparing Tcf7l2 against scramble siRNA treatment. Essential Cuffdiff command arguments were: --FDR 0.05 --library-type fr-unstranded --multi-read-correct --upper-quartile-norm --frag-bias-correct <rn4 genome fastas> <H4IIE-specific transcriptome>
ChIP-seq: Alignment to rn4 using Bowtie v0.12.8 with the following essential command line arguments: bowtie -n 1 -m 1 -k 1 --trim5 5 -l 36 --best
ChIP-seq: Conversion to sorted BAM format using samtools
ChIP-seq: Peak calling using MACS2 version 2.0.10.20130306 with the following essential command line arguments: macs2 callpeak –bw 100 –m 5 50 –keep-dup 1 --qvalue=0.01 --bgd
Genome_build: rn4
Supplementary_files_format_and_content: gtf: standard gtf as produced by Cuffcompare
Supplementary_files_format_and_content: differential expression: standard gene_exp.diff files produced by Cuffdiff
Supplementary_files_format_and_content: endocePeak: MACS2-native format that is otherwise like the standard ENCODE-style narrowPeak format except that the 5th column contains transformed p-values (-log10pvalue*10)

organism: Rattus norvegicus

source_name: H4IIE hepatocytes

growth_protocol: Low passage H4IIE cells were purchased from ATCC and used in the lab up to passage 30. The cells were routinely cultured in low glucose (5 mM) DMEM media supplemented with 10% fetal bovine serum in the absence of antibiotics and laboratory stocks tested negative for mycoplasma. After siRNA treatment, cells were immediately plated in 24-well plates in regular culture medium.

extract_protocol:
Chromatin was prepared from approximately 25 million siTCF7L2 or scramble electroporated cells by shearing with MNase using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology) to fragments ranging between 150 bp and 900 bp. Each aliquot was incubated overnight with either 5 ul of the anti-TCF7L2 antibody or 1 µl of the anti-HNF4α antibody. A sample consisting of 2% of the total input chromatin was removed from each and served as the sequencing control. Immunoprecipitated material was purified as described by the manufacturer (Cell Signaling Technology), except that replicate immunoprecipitations were pooled at the final Qiagen DNA purification step. Each sequenced sample represents a pool of 2 independent immunoprecipitations, each consisting of between 10 and 20 ug of chromatin.
ChIP-Seq libraries were generated using a combination of Illumina TruSeq adapters/indexes and a Kapa Library Preparation Kit (Kapa Biosystems, Woburn, MA, USA; cat # KK8200) with the following modifications. End-repair was performed on approximately 5 – 10 ng of ChIP sample. Qiagen Minelute purifications (Qiagen, Valencia, CA, USA) were utilized in between each step of adenylation and adapter ligation. Adapter ligation was carried out using stock Illumina adapters and Kapa T4 ligase at 30°C for 10 minutes, followed by a single DNA clean-up using homemade SPRI beads. The adapted material was minimally amplified for 5 cycles, using Illumina PCR primer cocktail and Kapa HiFi HotStart Polymerase ReadyMix (cat # KK2601), to convert y-shaped adapters to double-stranded DNA for more reliable electrophoresis, and then size selected on a 2% agarose gel before a final PCR amplification of 8 – 12 cycles and SPRI bead clean-up. Final libraries were checked for size using Bioanalyzer HS DNA or DNA 1000 chips.

treatment_protocol: Silencing of TCF7L2 mRNA and protein levels was achieved using 100 nM Dharmacon SmartPool siRNA (cat #), introduced via Neon electroporation, as described previously (PMID: 21901280). The experiment was performed in 1-3 replicates with separate electroporations being performed on a different passage of cells on a different date.

value: hepatocytes

value: siRNA - Scrambled

tag: antibody

value: HNF4A (Lot A-2, clone H1415, cat # PP-H1415-00, R&D Systems, Minneapolis, MN, USA)


title: ChIP-seq sample for siRNA knock-down of Tcf7l2: siRNA - Tcf7l2, 15 h, target protein - Hnf4a


title: ChIP-seq sample for siRNA knock-down of Tcf7l2: siRNA - Tcf7l2, 15 h, target protein - Tcf7l2, rep 2

value: siRNA -Tcf7l2

value: TCF7L2 (Lot 2, clone C48H11, cat # 2569, Cell Signaling Technology, Danvers, MA, USA)


title: L227_G04

description: single cell

value: C

value: Cooling

value: male


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title: L129_D10

description: single cell


title: ChIP-seq sample for siRNA knock-down of Tcf7l2: siRNA - Tcf7l2, 15 h, target protein - Tcf7l2, rep 1


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description: single cell


title: L129_D02

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title: L227_D06

description: single cell


title: ChIP-seq sample for siRNA knock-down of Tcf7l2: siRNA - Scrambled, 15 h, target protein - Tcf7l2, rep 2


title: L129_C12

description: single cell


title: ChIP-seq sample for siRNA knock-down of Tcf7l2: siRNA - Scrambled, 15 h, target protein - Tcf7l2, rep 1


title: Pooled Input chromatin

value: pooled


title: L227_D04

description: single cell


title: L129_C10

description: >1 cell


title: L227_D05

description: single cell


title: L129_C09

description: single cell


title: Pooled RNA-seq sample for H4IIE-specific transcriptome generation

extract_protocol:
RNA was extracted standard procedures.
Illumina sequencing libraries were prepared using the TruSeq RNA Sample Prep Kit V2.


title: L129_C08

description: single cell


title: L129_C07

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title: L227_D01

description: single cell


title: RNA-seq sample for timecourse of siRNA knock-down of Tcf7l2: siRNA - Tcf7l2, 96 h, rep 1


title: RNA-seq sample for timecourse of siRNA knock-down of Tcf7l2: siRNA - Tcf7l2, 48 h, rep 2


title: L227_C12

description: single cell


title: RNA-seq sample for timecourse of siRNA knock-down of Tcf7l2: siRNA - Tcf7l2, 18 h, rep 2


title: L129_C05

description: single cell


title: RNA-seq sample for timecourse of siRNA knock-down of Tcf7l2: siRNA - Tcf7l2, 48 h, rep 1


title: L227_C11

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title: L129_C04

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title: L227_C10

description: single cell


title: RNA-seq sample for timecourse of siRNA knock-down of Tcf7l2: siRNA - Tcf7l2, 18 h, rep 1


title: L129_C11

description: single cell


title: L227_D03

description: single cell


title: RNA-seq sample for timecourse of siRNA knock-down of Tcf7l2: siRNA - Tcf7l2, 15 h, rep 2


title: L129_C03

description: single cell


title: RNA-seq sample for timecourse of siRNA knock-down of Tcf7l2: siRNA - Tcf7l2, 96 h, rep 2


title: L129_C02

description: single cell