title: opossum1_RS_K27me3_ChIP

data_processing:
Image analysis and base calling was done using the standard Illumina pipeline for HiSeq2500.
Data was quality-filtered using fastq_quality_filter from the FASTX toolkit with the following parameters: -q 20 -p 80. For libraries with >50% adapter reads (one library total: human_PS_K4me3_ChIP), reads containing only adapter sequence were discarded using fastx_clipper.
For ChIP-seq, alignment was done using Bowtie 1.1.1 with the following parameters: -k 1 -m 1 -n 1 -l 40.  For RNA-seq, alignment was done using TopHat 2.0 with default settings, with Ensembl transcripts as a reference (-G parameter).
For ChIP-seq, peaks were called using MACS 1.4 at p < 10^-5 (-p 1e-5), with default parameters.
For ChIP-seq, raw counts in the +/- 2kb intervals surrounding the transcription start sites were calculated using htseq-count with the following settings: -m intersection-nonempty -s no.
genome build: monDom5
processed data files format and content: All files are tab-delimited text (.txt).  For RNA-seq, FPKM is reported for each Ensembl (build 75) transcript and gene.  For ChIP-seq, raw counts normalized to (aligned, non-duplicated) library size are reported for the +/- 2kb region surrounding each Ensembl (build 75) transcript.

growth_protocol: Testes from Monodelphis domestica were obtained from animals culled from a colony maintained at the Texas Biomedical Research Institute.

extract_protocol:
Populations of pachytene spermatocytes and round spermatids (purity >95%) were recovered using a StaPut gradient as previously described (Bellve et al, 1993).  Cells were washed once in PBS and then split into two aliquots.  One aliquot (for ChIP) was fixed in 1% formaldehyde then quenched with 2.5M glycine, while the second (for RNA) was left unfixed.  Both fixed and unfixed aliquots were snap frozen in liquid nitrogen, then stored at -80C. ChIP was performed as previously described (Lesch et al 2013).
ChIP-seq libraries were prepared using a TruSeq ChIP sample prep kit (Illumina) according to the manufacturer’s instructions, except that size selection was performed after instead of before PCR amplification.


title: opossum1_RS_input_for_ChIP

status: Public on May 07 2016

type: SRA

city: New Haven

first: Bluma

middle: J

last: Lesch

state: CT

address: 333 Cedar Street

department: Genetics

country: USA

institute: Yale University

library_source: genomic

molecule: genomic DNA

organism: Monodelphis domestica

source_name: adult testis

tag: tissue

value: testis

tag: cell type

value: round spermatids

tag: chip antibody


title: opossum1_RS_K4me3_ChIP

value: H3K4me3 (abcam, #ab8580)


title: opossum1_PS_RNA

library_source: transcriptomic

data_processing:
Image analysis and base calling was done using the standard Illumina pipeline for HiSeq2500.
Data was quality-filtered using fastq_quality_filter from the FASTX toolkit with the following parameters: -q 20 -p 80. For libraries with >50% adapter reads (one library total: human_PS_K4me3_ChIP), reads containing only adapter sequence were discarded using fastx_clipper.
For RNA-seq, FPKM values were generated using Cufflinks 2.2 with default parameters, except that a transcript gtf was supplied (-G option), and average fragment length of the library was supplied under the -m flag.
genome build: monDom5
processed data files format and content: All files are tab-delimited text (.txt).  For RNA-seq, FPKM is reported for each Ensembl (build 75) transcript and gene.  For ChIP-seq, raw counts normalized to (aligned, non-duplicated) library size are reported for the +/- 2kb region surrounding each Ensembl (build 75) gene.

molecule: total RNA

extract_protocol:
Populations of pachytene spermatocytes and round spermatids (purity >95%) were recovered using a StaPut gradient as previously described (Bellve et al, 1993).  Cells were washed once in PBS and then split into two aliquots.  One aliquot (for ChIP) was fixed in 1% formaldehyde then quenched with 2.5M glycine, while the second (for RNA) was left unfixed.  Both fixed and unfixed aliquots were snap frozen in liquid nitrogen, then stored at -80C. Unfixed aliquots of sorted cells were thawed on ice, and RNA isolation was performed using the RNEasy Mini kit according to the manufacturer’s instructions.  Genomic DNA was removed using gDNA eliminator columns supplied with the kit.
RNA-seq libraries were prepared using an Apollo 324 library prep instrument with supplied reagents (Integenx).

value: pachytene spermatocytes


title: opossum1_PS_input_for_ChIP


title: opossum2_PS_K27me3_ChIP

description: Testes from Monodelphis domestica were obtained from animals culled from a colony maintained at the Texas Biomedical Research Institute.

data_processing:
Image analysis and base calling was done using the standard Illumina pipeline for HiSeq2500.
Data was quality-filtered using fastq_quality_filter from the FASTX toolkit with the following parameters: -q 20 -p 80.
For ChIP-seq, alignment was done using Bowtie 1.1.1 with the following parameters: -k 1 -m 1 -n 1 -l 40.  For RNA-seq, alignment was done using TopHat 2.0 with default settings, with Ensembl transcripts as a reference (-G parameter).
For ChIP-seq, peaks were called using MACS 1.4 at p < 10^-5 (-p 1e-5), with default parameters.
For ChIP-seq, raw counts in the +/- 2kb intervals surrounding the transcription start sites for each transcript were calculated using htseq-count with the following settings: -m intersection-nonempty -s no.
genome build: monDom5
processed data files format and content: All files are tab-delimited text (.txt).   For ChIP-seq, raw counts normalized to (aligned, non-duplicated) library size are reported for the +/- 2kb region surrounding each Ensembl (build 75) gene.

extract_protocol:
Populations of pachytene spermatocytes and round spermatids (purity 89-90%) were recovered using a StaPut gradient as previously described (Bellve et al, 1993).  Cells were washed once in PBS and then split into two aliquots.  One aliquot (for ChIP) was fixed in 1% formaldehyde then quenched with 2.5M glycine, while the second (for RNA) was left unfixed.  Both fixed and unfixed aliquots were snap frozen in liquid nitrogen, then stored at -80C. ChIP was performed as previously described (Lesch et al 2013).
ChIP-seq libraries were prepared using a TruSeq ChIP sample prep kit (Illumina) according to the manufacturerÕs instructions, except that size selection was performed after instead of before PCR amplification.


title: opossum1_RS_RNA


title: CS_cDNA_KD2

city: Leuven

first: Zeynep

last: Kalender Atak

address: Herestraat 49

country: Belgium

institute: KULeuven

description: cDNA

data_processing:
Library strategy: CHEQ-Seq
Files were cleaned and trimmed where needed.
Reads were mapped to the hg19 genome using STAR.
The sensitive-local setting for Bowtie2 was used to correct for a high percentage of mismatches at the start of a read, resulting in the removal of the first five base pairs for each read.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: *counts.txt: Counts are raw and were generated using HTseq counts.

organism: Homo sapiens

source_name: MCF7, p53 knockdown, non-stimulated

growth_protocol: Cells were grown at 37°C to 85% confluency before harvest. Medium used was Ham's F-10 nutrient mix supplemented with FBS and l-alanyl-glutamine.

extract_protocol:
DNA was extracted with a plasmid extraction protocol from Qiagen; RNA was extracted using the RNeasy protocol from Qiagen.
Custom library prep.

treatment_protocol: p53 stimulation with Nutlin-3a or stable knockdown of p53.

tag: cell line

tag: genotype/variation

value: stable knockdown of p53

tag: treatment

tag: capturing system

value: MYbaits custom capturing system


title: opossum2_RS_input_for_ChIP


title: CS_input

description:
Plasmid DNA library input.
HiSeq sequencing of the plasmid library that was also transfected.

source_name: MCF7, input


title: bull_RS_input_for_ChIP

data_processing:
Image analysis and base calling was done using the standard Illumina pipeline for HiSeq2500.
Data was quality-filtered using fastq_quality_filter from the FASTX toolkit with the following parameters: -q 20 -p 80. For libraries with >50% adapter reads (one library total: human_PS_K4me3_ChIP), reads containing only adapter sequence were discarded using fastx_clipper.
For ChIP-seq, alignment was done using Bowtie 1.1.1 with the following parameters: -k 1 -m 1 -n 1 -l 40.  For RNA-seq, alignment was done using TopHat 2.0 with default settings, with Ensembl transcripts as a reference (-G parameter).
For ChIP-seq, peaks were called using MACS 1.4 at p < 10^-5 (-p 1e-5), with default parameters.
For ChIP-seq, raw counts in the +/- 2kb intervals surrounding the transcription start sites were calculated using htseq-count with the following settings: -m intersection-nonempty -s no.
genome build: bosTau7
processed data files format and content: All files are tab-delimited text (.txt).  For RNA-seq, FPKM is reported for each Ensembl (build 75) transcript and gene.  For ChIP-seq, raw counts normalized to (aligned, non-duplicated) library size are reported for the +/- 2kb region surrounding each Ensembl (build 75) transcript.

organism: Bos taurus

growth_protocol: Testes from a bull were obtained as abattoir material.


title: bull_RS_RNA

data_processing:
Image analysis and base calling was done using the standard Illumina pipeline for HiSeq2500.
Data was quality-filtered using fastq_quality_filter from the FASTX toolkit with the following parameters: -q 20 -p 80. For libraries with >50% adapter reads (one library total: human_PS_K4me3_ChIP), reads containing only adapter sequence were discarded using fastx_clipper.
For RNA-seq, FPKM values were generated using Cufflinks 2.2 with default parameters, except that a transcript gtf was supplied (-G option), and average fragment length of the library was supplied under the -m flag.
genome build: bosTau7
processed data files format and content: All files are tab-delimited text (.txt).  For RNA-seq, FPKM is reported for each Ensembl (build 75) transcript and gene.  For ChIP-seq, raw counts normalized to (aligned, non-duplicated) library size are reported for the +/- 2kb region surrounding each Ensembl (build 75) gene.


title: bull_PS_RNA


title: bull_RS_K4me3_ChIP


title: opossum1_PS_K27me3_ChIP


title: bull_PS_input_for_ChIP


title: bull_PS_K4me3_ChIP


title: bull_PS_K27me3_ChIP


title: rhesus2_RS_RNA

data_processing:
Image analysis and base calling was done using the standard Illumina pipeline for HiSeq2500.
Data was quality-filtered using fastq_quality_filter from the FASTX toolkit with the following parameters: -q 20 -p 80. For libraries with >50% adapter reads (one library total: human_PS_K4me3_ChIP), reads containing only adapter sequence were discarded using fastx_clipper.
For RNA-seq, FPKM values were generated using Cufflinks 2.2 with default parameters, except that a transcript gtf was supplied (-G option), and average fragment length of the library was supplied under the -m flag.
genome build: rheMac2
processed data files format and content: All files are tab-delimited text (.txt).  For RNA-seq, FPKM is reported for each Ensembl (build 75) transcript and gene.  For ChIP-seq, raw counts normalized to (aligned, non-duplicated) library size are reported for the +/- 2kb region surrounding each Ensembl (build 75) gene.

organism: Macaca mulatta

growth_protocol: Testes from were obtained from a rhesus monkey undergoing necropsy for other purposes at the Texas Biomedical Research Institute.


title: rhesus2_RS_K4me3_ChIP

data_processing:
Image analysis and base calling was done using the standard Illumina pipeline for HiSeq2500.
Data was quality-filtered using fastq_quality_filter from the FASTX toolkit with the following parameters: -q 20 -p 80. For libraries with >50% adapter reads (one library total: human_PS_K4me3_ChIP), reads containing only adapter sequence were discarded using fastx_clipper.
For ChIP-seq, alignment was done using Bowtie 1.1.1 with the following parameters: -k 1 -m 1 -n 1 -l 40.  For RNA-seq, alignment was done using TopHat 2.0 with default settings, with Ensembl transcripts as a reference (-G parameter).
For ChIP-seq, peaks were called using MACS 1.4 at p < 10^-5 (-p 1e-5), with default parameters.
For ChIP-seq, raw counts in the +/- 2kb intervals surrounding the transcription start sites were calculated using htseq-count with the following settings: -m intersection-nonempty -s no.
genome build: rheMac2
processed data files format and content: All files are tab-delimited text (.txt).  For RNA-seq, FPKM is reported for each Ensembl (build 75) transcript and gene.  For ChIP-seq, raw counts normalized to (aligned, non-duplicated) library size are reported for the +/- 2kb region surrounding each Ensembl (build 75) transcript.


title: rhesus2_RS_K27me3_ChIP


title: opossum1_PS_K4me3_ChIP


title: CS_cDNA_KD1

description: cDNA


title: rhesus2_PS_K4me3_ChIP


title: rhesus2_PS_input_for_ChIP


title: rhesus2_PS_K27me3_ChIP


title: rhesus1_RS_RNA


title: rhesus2_RS_input_for_ChIP


title: rhesus1_RS_K4me3_ChIP


title: rhesus1_RS_K27me3_ChIP


title: cell 78

city: Tarrytown

first: Ruoyu

last: Zhang

address: 777 Old Saw Mill River Road

institute: Regeneron

data_processing:
Library strategy: DNA-Seq
Raw sequencing reads were trimmed by Trimmomatic
Cleaned reads were then mapped to hg19 human reference sequence with rCRS mitochondrial reference genome by bowtie2
PCR duplicates were removed with Picard
mtDNA heteroplasmies were called by customer scripts
Genome_build: hg19,rCRS

source_name: cell standard process method

extract_protocol:
In standard method, DNA was extracted by DNeasy Blood and Tissue Kit (No. 69504; Qiagen); in optimized method, DNA was extracted by QIAamp DSP DNA Blood Mini Kit (No. 61104; Qiagen). White blood cell DNA was extracted by DNeasy Blood and Tissue Kit (No. 69504; Qiagen).
In standard method, libraries were prepared by standard library preparation protocol. In optimized method, libraries were prepared by KAPA Hyper Prep Kits (No. KK8502; Kapa). Cell DNA libraries were prepared by by Nextera XT DNA library preparation kit (Illumina, FC-131-1024).

value: white blood cell


title: cell 69


title: rhesus1_PS_RNA


title: rhesus1_PS_input_for_ChIP


title: rhesus1_RS_input_for_ChIP


title: bull_RS_K27me3_ChIP


title: rhesus1_PS_K27me3_ChIP


title: rhesus1_PS_K4me3_ChIP


title: mouse sperm head RNA-Seq rep2

city: reno

first: Wei

last: Yan

state: NV

address: 1664 N. Virginia Street

department: Physiology

institute: University of Nevada

data_processing:
Torrent suite
RNA-Seq: Sequenced reads were mapped to (mm10, mouse; rn5, rat; oryCun2, rabbit; hg19, human) whole genome using TOPHAT; sncRNA-Seq: Sequenced reads were matched to sncRNA sequences via Sequery and then mapped to (mm10, mouse; rn5, rat; oryCun2, rabbit; hg19, human) via Bowtie.
RNA-Seq: Fragments Per Kilobase of exon per Megabase of library size (RPKM) were calculated and isoforms were assembled by using a protocol from Cufflinks; sncRNA-Seq: reads per 100,000 aligned reads (RPK) were calculated
Genome_build: mm10, mouse; hg19, human; oryCun2, rabbit; rn5, rat
Supplementary_files_format_and_content: Averaged, normalized expression values are provided for the RNA-Seq (RPKM) and sncRNA-Seq (RPK) samples. The processed data table is organized by gene name ("Gene"), species of origin ("Species"), sample type, i.e., total sperm or sperm head ("Sample"), class of RNA ("Class"), normalized average expression ("Expression")

organism: Mus musculus

source_name: mouse sperm head RNA-Seq

extract_protocol:
Total RNA was extracted from sperm samples using the mirVana total RNA extraction protocol. Human and rabbit sperm samples were pipeted up and down during lysis; mouse sperm samples were homogenized for one minute during lysis; rat samples were homogenized for 90 seconds and subsequently incubated at 65C for five minutes during lysis.
RNA libraries were prepared for sequencing using standard Ion Torrent protocols

tag: strain

tag: age

value: adult

tag: genotype

value: wild type

value: sperm head


title: mouse total sperm RNA-Seq rep1

source_name: mouse total sperm RNA-Seq

value: total sperm


title: mouse sperm head RNA-Seq rep1


title: mouse total sperm RNA-Seq rep2


title: cell 93


title: mouse total sperm sncRNA-Seq rep2

source_name: mouse total sperm sncRNA-Seq


title: chicken_RS_RNA

data_processing:
Image analysis and base calling was done using the standard Illumina pipeline for HiSeq2500.
Data was quality-filtered using fastq_quality_filter from the FASTX toolkit with the following parameters: -q 20 -p 80. For libraries with >50% adapter reads (one library total: human_PS_K4me3_ChIP), reads containing only adapter sequence were discarded using fastx_clipper.
For RNA-seq, FPKM values were generated using Cufflinks 2.2 with default parameters, except that a transcript gtf was supplied (-G option), and average fragment length of the library was supplied under the -m flag.
genome build: galGal4
processed data files format and content: All files are tab-delimited text (.txt).  For RNA-seq, FPKM is reported for each Ensembl (build 75) transcript and gene.  For ChIP-seq, raw counts normalized to (aligned, non-duplicated) library size are reported for the +/- 2kb region surrounding each Ensembl (build 75) gene.

organism: Gallus gallus

growth_protocol: Testes from three roosters were obtained as abattoir material, and tissue was pooled.

extract_protocol:
Populations of pachytene spermatocytes and round spermatids (purity >95%) were recovered using a StaPut gradient as previously described (Bellve et al, 1993).  Cells were washed once in PBS and then split into two aliquots.  One aliquot (for ChIP) was fixed in 1% formaldehyde then quenched with 2.5M glycine, while the second (for RNA) was left unfixed.  Both fixed and unfixed aliquots were snap frozen in liquid nitrogen, then stored at -80C.  Unfixed aliquots of sorted cells were thawed on ice, and RNA isolation was performed using the RNEasy Mini kit according to the manufacturer’s instructions.  Genomic DNA was removed using gDNA eliminator columns supplied with the kit.
RNA-seq libraries were prepared using an Apollo 324 library prep instrument with supplied reagents (Integenx).


title: mouse total sperm sncRNA-Seq rep1


title: A. niger+T. reesei-straw media replicate flask-C

city: Nottingham

first: David

middle: B

last: Archer

address: University Park

department: School of Life Sciences

country: United Kingdom

institute: University of Nottingham

description:
BP-33
A.niger_FPKMs.txt
T.reesei_FPKMs.txt

data_processing:
A filtering pipeline was used to filter reads with low sequencing quality score as well as reads aligned to adaptor sequences. First, raw reads were trimmed against adaptors using Scythe (https://github.com/vsbuffalo/scythe). Then reads were quality trimmed using Sickle (https://github.com/najoshi/sickle).
For all samples, reads that passed the filters were mapped using the tophat mapping tool (https://ccb.jhu.edu/software/tophat/index.shtml) onto a composite reference genome that consisted of the genomes of each of the three species.
The composite genome was made by concatenating into a single file the fasta files made of the sequences for the chromosomes from Aspni7, Trire2 and Penchwisc1_1 (where repeat regions had been masked). The use of this composite genome facilitated the exclusion from subsequent analysis of reads that were not unique to a particular species as these reads would not be uniquely mapped in the composite genome.
Uniquely mapped read counts (at the MAPQ20 threshold) for each gene were calculated using 'htseq-count' (http://www-huber.embl.de/users/anders/HTSeq/doc/count.html). Read counts were calculated for each species separately using a genome annotation file for each species that contained the known gene exon co-ordinates for the genes for that species. The genome annotation files (downloaded from the JGI) used were as follows: Aspni7 – Aspni7_GeneCatalog_genes_20131226.gff, Trire2 – TreeseiV2_FrozenGeneCatalog20081022.gff and Penchwisc1_1 – PenchWisc1_1_GeneCatalog_genes_20140114.gff.
FPKM normalised read counts were defined originally by Mortazavi et al. (2008). In our study, an FPKM value for a gene from a sample is the number of uniquely mapped (to the composite genome) fragments (counts) per kilobase of gene model per million uniquely mapped (to the composite genome) fragments (counts) onto gene models from that species.
Genome_build: Sequences from the JGI for Aspni7, Trire2 and Penchwisc1_1 (where repeat regions had been masked).
Supplementary_files_format_and_content: tab delimited text files with FPKM values for each sample.
Supplementary_files_format_and_content: FASTA file with nucleotide sequeces of contigs from each of the three species that was used as the reference to map reads.

organism:
Aspergillus niger
Trichoderma reesei

source_name: fungal mycelia from shake-flask culture

growth_protocol: Please see the publication for this dataset from Fungal Genetics and Biology with is available as 'open access'.

extract_protocol:
Mycelia were ground under liquid nitrogen using a mortar and pestle, then the RNA was purified using the Plant/Fungi total RNA Purification Kit (Norgen Biotek, Canada) including the on-column DNase treatment step.
Total RNA was measured using Qubit RNA BR assay kit (Life technologies, Q10210). 1 µg of Total RNA was used for enrichment of mRNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490). Illumina stranded whole transcriptome sequencing libraries were prepared using NEBNext Ultra Directional RNA library prep kit for Illumina (NEB, E7420S). Library QC was performed using bioanalyser HS kit (Agilent biotechnologies, 5067-4626). Libraries were quantified using qPCR (Kapa Biosystems, KK4824), pooled at desired concentrations, denatured and loaded for sequencing according to manufacturer’s instructions. Sequencing was done on Illumina NextSeq500 sequencing platform using 2 high output runs to generate 2 x 75 bp reads. Sufficient sequencing was done to obtain ~30 M reads from each monoculture replicate and ~ 100 M reads from each mixed culture replicate. Note: For consistency and to avoid bias, all 30 samples were pooled for running on a lane which resulted in a read output for each sample from each of the four lanes from each of the two runs with the exception of sample BP-23 which was only run once. This would generate 480 sequencing files ((read files (total 2) from each sample (total 30) from each lane (total 4) for each run (total 2)) = 2 x 30 x 4 x 2 = 480) except only 472 files were generated as  sample BP-23  was only run in run 1.

tag: mixed culture

value: Aspergillus niger and Trichoderma reesei


title: rhesus2_PS_RNA


title: A. niger+T. reesei-straw media replicate flask-B

description:
BP-32
A.niger_FPKMs.txt
T.reesei_FPKMs.txt

treatment_protocol: Please see the publication for this dataset from Fungal Genetics and Biology with is available as 'open access'.


title: chicken_RS_input_for_ChIP

data_processing:
Image analysis and base calling was done using the standard Illumina pipeline for HiSeq2500.
Data was quality-filtered using fastq_quality_filter from the FASTX toolkit with the following parameters: -q 20 -p 80. For libraries with >50% adapter reads (one library total: human_PS_K4me3_ChIP), reads containing only adapter sequence were discarded using fastx_clipper.
For ChIP-seq, alignment was done using Bowtie 1.1.1 with the following parameters: -k 1 -m 1 -n 1 -l 40.  For RNA-seq, alignment was done using TopHat 2.0 with default settings, with Ensembl transcripts as a reference (-G parameter).
For ChIP-seq, peaks were called using MACS 1.4 at p < 10^-5 (-p 1e-5), with default parameters.
For ChIP-seq, raw counts in the +/- 2kb intervals surrounding the transcription start sites were calculated using htseq-count with the following settings: -m intersection-nonempty -s no.
genome build: galGal4
processed data files format and content: All files are tab-delimited text (.txt).  For RNA-seq, FPKM is reported for each Ensembl (build 75) transcript and gene.  For ChIP-seq, raw counts normalized to (aligned, non-duplicated) library size are reported for the +/- 2kb region surrounding each Ensembl (build 75) transcript.

extract_protocol:
Populations of pachytene spermatocytes and round spermatids (purity >95%) were recovered using a StaPut gradient as previously described (Bellve et al, 1993).  Cells were washed once in PBS and then split into two aliquots.  One aliquot (for ChIP) was fixed in 1% formaldehyde then quenched with 2.5M glycine, while the second (for RNA) was left unfixed.  Both fixed and unfixed aliquots were snap frozen in liquid nitrogen, then stored at -80C.  ChIP was performed as previously described (Lesch et al 2013).
ChIP-seq libraries were prepared using a TruSeq ChIP sample prep kit (Illumina) according to the manufacturer’s instructions, except that size selection was performed after instead of before PCR amplification.


title: A. niger+T. reesei-straw media replicate flask-A

description:
BP-31
A.niger_FPKMs.txt
T.reesei_FPKMs.txt


title: chicken_PS_RNA


title: T. reesei-straw media replicate flask-C

description:
BP-27
T.reesei_FPKMs.txt

organism: Trichoderma reesei


title: chicken_RS_K27me3_ChIP


title: chicken_RS_K4me3_ChIP


title: chicken_PS_input_for_ChIP


title: T. reesei-straw media replicate flask-B

description:
BP-26
T.reesei_FPKMs.txt


title: T. reesei-straw media replicate flask-A

description:
BP-25
T.reesei_FPKMs.txt


title: rat total sperm RNA-Seq rep2

organism: Rattus norvegicus

source_name: rat total sperm RNA-Seq

value: Hsd:Sprague Dawley


title: rat total sperm RNA-Seq rep3


title: T. reesei-glucose media replicate flask-C

description:
BP-6
T.reesei_FPKMs.txt


title: rat total sperm sncRNA-Seq rep3

source_name: rat total sperm sncRNA-Seq


title: T. reesei-glucose media replicate flask-B

description:
BP-5
T.reesei_FPKMs.txt


title: rat total sperm RNA-Seq rep1


title: mouse2_RS_input_for_ChIP

data_processing:
Image analysis and base calling was done using the standard Illumina pipeline for HiSeq2500.
Data was quality-filtered using fastq_quality_filter from the FASTX toolkit with the following parameters: -q 20 -p 80. For libraries with >50% adapter reads (one library total: human_PS_K4me3_ChIP), reads containing only adapter sequence were discarded using fastx_clipper.
For ChIP-seq, alignment was done using Bowtie 1.1.1 with the following parameters: -k 1 -m 1 -n 1 -l 40.  For RNA-seq, alignment was done using TopHat 2.0 with default settings, with Ensembl transcripts as a reference (-G parameter).
For ChIP-seq, peaks were called using MACS 1.4 at p < 10^-5 (-p 1e-5), with default parameters.
For ChIP-seq, raw counts in the +/- 2kb intervals surrounding the transcription start sites were calculated using htseq-count with the following settings: -m intersection-nonempty -s no.
genome build: mm10
processed data files format and content: All files are tab-delimited text (.txt).  For RNA-seq, FPKM is reported for each Ensembl (build 75) transcript and gene.  For ChIP-seq, raw counts normalized to (aligned, non-duplicated) library size are reported for the +/- 2kb region surrounding each Ensembl (build 75) transcript.

growth_protocol: Testes were obtained from adult CD1 wild type male mice (Charles River Laboratories).

extract_protocol:
Populations of pachytene spermatocytes and round spermatids were recovered using a StaPut gradient as previously described (Bellve et al, 1993).  All isolated fractions were at least 95% pure.  Cells were washed once in PBS and then split into two aliquots.  One aliquot (for ChIP) was fixed in 1% formaldehyde then quenched with 2.5M glycine, while the second (for RNA) was left unfixed.  Both fixed and unfixed aliquots were snap frozen in liquid nitrogen, then stored at -80C.  ChIP was performed as previously described (Lesch et al 2013).
ChIP-seq libraries were prepared using a TruSeq ChIP sample prep kit (Illumina) according to the manufacturer’s instructions, except that size selection was performed after instead of before PCR amplification.


title: rat total sperm sncRNA-Seq rep1


title: T. reesei-glucose media replicate flask-A

description:
BP-4
T.reesei_FPKMs.txt


title: rat total sperm sncRNA-Seq rep2


title: human total sperm sncRNA-Seq rep2

source_name: human total sperm sncRNA-Seq


title: rabbit sperm head RNA-Seq rep2

organism: Oryctolagus cuniculus

source_name: rabbit sperm head RNA-Seq


title: mouse2_RS_K4me3_ChIP


title: mouse sperm head sncRNA-Seq rep2

source_name: mouse sperm head sncRNA-Seq


title: human total sperm sncRNA-Seq rep3


title: rabbit sperm head RNA-Seq rep3


title: chicken_PS_K27me3_ChIP


title: mouse2_PS_RNA

data_processing:
Image analysis and base calling was done using the standard Illumina pipeline for HiSeq2500.
Data was quality-filtered using fastq_quality_filter from the FASTX toolkit with the following parameters: -q 20 -p 80. For libraries with >50% adapter reads (one library total: human_PS_K4me3_ChIP), reads containing only adapter sequence were discarded using fastx_clipper.
For RNA-seq, FPKM values were generated using Cufflinks 2.2 with default parameters, except that a transcript gtf was supplied (-G option), and average fragment length of the library was supplied under the -m flag.
genome build: mm10
processed data files format and content: All files are tab-delimited text (.txt).  For RNA-seq, FPKM is reported for each Ensembl (build 75) transcript and gene.  For ChIP-seq, raw counts normalized to (aligned, non-duplicated) library size are reported for the +/- 2kb region surrounding each Ensembl (build 75) gene.

extract_protocol:
Populations of pachytene spermatocytes and round spermatids were recovered using a StaPut gradient as previously described (Bellve et al, 1993).  All isolated fractions were at least 95% pure.  Cells were washed once in PBS and then split into two aliquots.  One aliquot (for ChIP) was fixed in 1% formaldehyde then quenched with 2.5M glycine, while the second (for RNA) was left unfixed.  Both fixed and unfixed aliquots were snap frozen in liquid nitrogen, then stored at -80C.  Unfixed aliquots of sorted cells were thawed on ice, and RNA isolation was performed using the RNEasy Mini kit according to the manufacturer’s instructions.  Genomic DNA was removed using gDNA eliminator columns supplied with the kit.
RNA-seq libraries were prepared using an Apollo 324 library prep instrument with supplied reagents (Integenx).


title: rabbit sperm head RNA-Seq rep1


title: mouse sperm head sncRNA-Seq rep1


title: T. reesei+P. chrysogenum-straw media replicate flask-C

description:
BP-36
P.chrysogenum_FPKMs.txt
T.reesei_FPKMs.txt

organism:
Penicillium chrysogenum
Trichoderma reesei

value: QM6a, Wisconsin54-1255

value: Trichoderma reesei and Penicillium chrysogenum


title: rabbit total sperm RNA-Seq rep3

source_name: rabbit total sperm RNA-Seq


title: chicken_PS_K4me3_ChIP


title: T. reesei+P. chrysogenum-straw media replicate flask-B

description:
BP-35
P.chrysogenum_FPKMs.txt
T.reesei_FPKMs.txt


title: mouse2_PS_input_for_ChIP


title: mouse2_RS_RNA


title: mouse2_PS_K27me3_ChIP


title: mouse2_PS_K4me3_ChIP


title: T. reesei+P. chrysogenum-straw media replicate flask-A

description:
BP-34
P.chrysogenum_FPKMs.txt
T.reesei_FPKMs.txt


title: mouse2_RS_K27me3_ChIP


title: A. niger-straw media replicate flask-C

description:
BP-24
A.niger_FPKMs.txt

organism: Aspergillus niger


title: A. niger+T. reesei+P. chrysogenum-straw media replicate flask-C

description:
BP-42
A.niger_FPKMs.txt
P.chrysogenum_FPKMs.txt
T.reesei_FPKMs.txt

organism:
Aspergillus niger
Penicillium chrysogenum
Trichoderma reesei

value: N402, QM6a, Wisconsin54-1255

value: Aspergillus niger, Trichoderma reesei and Penicillium chrysogenum


title: rabbit total sperm RNA-Seq rep1


title: human total sperm sncRNA-Seq rep1


title: P. chrysogenum-straw media replicate flask-C

description:
BP-30
P.chrysogenum_FPKMs.txt

organism: Penicillium chrysogenum


title: A. niger-straw media replicate flask-B

description:
BP-23
A.niger_FPKMs.txt


title: rabbit sperm head sncRNA-Seq rep2

source_name: rabbit sperm head sncRNA-Seq


title: P. chrysogenum-straw media replicate flask-B

description:
BP-29
P.chrysogenum_FPKMs.txt


title: A. niger+T. reesei+P. chrysogenum-straw media replicate flask-B

description:
BP-41
A.niger_FPKMs.txt
P.chrysogenum_FPKMs.txt
T.reesei_FPKMs.txt


title: rabbit sperm head sncRNA-Seq rep3


title: A. niger-straw media replicate flask-A

description:
BP-22
A.niger_FPKMs.txt


title: rabbit sperm head sncRNA-Seq rep1


title: P. chrysogenum-straw media replicate flask-A

description:
BP-28
P.chrysogenum_FPKMs.txt


title: A. niger+T. reesei+P. chrysogenum-straw media replicate flask-A

description:
BP-40
A.niger_FPKMs.txt
P.chrysogenum_FPKMs.txt
T.reesei_FPKMs.txt


title: A. niger-glucose media replicate flask-C

description:
BP-3
A.niger_FPKMs.txt


title: rabbit total sperm RNA-Seq rep2


title: A. niger-glucose media replicate flask-B

description:
BP-2
A.niger_FPKMs.txt


title: P. chrysogenum-glucose media replicate flask-B

description:
BP-8
P.chrysogenum_FPKMs.txt


title: rabbit total sperm sncRNA-Seq rep3

source_name: rabbit total sperm sncRNA-Seq


title: P. chrysogenum-glucose media replicate flask-A

description:
BP-7
P.chrysogenum_FPKMs.txt


title: A. niger+P. chrysogenum-straw media replicate flask-B

description:
BP-38
A.niger_FPKMs.txt
P.chrysogenum_FPKMs.txt

organism:
Aspergillus niger
Penicillium chrysogenum

value: Aspergillus niger and Penicillium chrysogenum


title: rabbit total sperm sncRNA-Seq rep2


title: A. niger+P. chrysogenum-straw media replicate flask-C

description:
BP-39
A.niger_FPKMs.txt
P.chrysogenum_FPKMs.txt


title: A. niger-glucose media replicate flask-A

description:
BP-1
A.niger_FPKMs.txt


title: rabbit total sperm sncRNA-Seq rep1


title: A. niger+P. chrysogenum-straw media replicate flask-A

description:
BP-37
A.niger_FPKMs.txt
P.chrysogenum_FPKMs.txt


title: DNA methylation-Ebf1(-/-)::EBF1ΔC

status: Public on May 06 2016

city: Freiburg

first: Senthilkumar

last: Ramamoorthy

address: Stübeweg 51

department: Cellular & Molecular Immunology

country: Germany

institute: Max Planck Institute of Immunobiology and Epigenetics

description: Bisulfite-Seq profile of Ebf1(-/-)::EBF1ΔC

data_processing:
Basecalls and fastq files were generated using Illumina bcl2fastq 1.8.4 (RTA 1.18.64)
The reads were aligned to the mm9 genome using the R package QuasR (version 1.2.2) and positions covered by at least 5 reads were used.
Genome_build: mm9
Supplementary_files_format_and_content: The data contains information about the methylation status of each CpG in the mouse mm9 genome that is covered by at least one read. The file format is chr (chromosome), position, nTot and nMeth, where position is the coordinate of the cytosine in the CpG on the plus strand (the counts have been pooled from two strands), nTot is the total number of reads overlapping the cytosine and nMeth the total number of reads without cytosine to thymine conversion (reflecting methylated cytosines).

source_name: Ebf1−/− c-Kit+ fetal liver cells, pmys-EBF1ΔC-IRES-GFP

growth_protocol: Single cell suspensions of freshly isolated Ebf1−/− fetal liver cells were stained with biotinylated c-Kit and c-Kit positive cells were enriched on an AutoMACS separator and subsequently plated on OP9 feeder cells in OptiMEM medium, supplemented with 4% FCS, 1% PSG, 50 μM β-mercaptoethanol, 5 ng/ml interleukin 7 (IL-7), 10 ng/ml Flt3 ligand (Flt3L) and 10 ng/ml stem cell factor (SCF).

extract_protocol:
DNA and was extracted with phenol-chloroformisoamyl alcohol and RNA was removed by treatment with RNase A. Subsequently, DNA was sheared into pieces of average 600bp with a Bioruptor sonifier.
3 μg of sonified genomic DNA was mixed with 10 ng of unmethylated, sonified Lambda phage genome and 10ng of in vitro methylated, sonified PhiX 174genome as spike in controls. The DNA was end repaired, 3’ adenylated, and ligated to methylated paired-end adaptors. Adapter-ligated DNA of 350-400bp were gel purified and DNA was converted with sodium bisulfite.The bisulfite-converted adapter-ligated DNA molecules were enriched using 10 cycles of PCR with PfuTurboCx Hotstart DNA polymerase. The reaction was purified to separate the library from adapter-adapter ligation products.

treatment_protocol: Retroviral transductions with pMys-EBF1wt-IRES-GFP, pMys-EBF1ΔC-IRES-GFP or pMys-IRES-GFP (vector) retrovirus were performed. 24h after transduction, cells were sorted as GFP+ cells and re-plated on OP9 cells.

value: Ebf1-/- c-Kit+ fetal liver cells

tag: transfection construct

value: fetal liver


title: Input-Ebf1(-/-)::EBF1ΔC

description: Input DNA of Ebf1(-/-)::EBF1ΔC cells

data_processing:
Basecalling and fastq files generation was performed by using Illumina bcl2fastq 1.8.4 (RTA 1.18.64)
The reads were aligned to the mm9 genome assembly using bowtie2 with default configurations
Duplicated reads were filtered and blacklisted regions were removed using samtools and Picard.
Peaks were identified using MACS2 (v2.1.0.20150731) with q-value cutoff 0.01 and further filtered for p-value 10-10 and fold change 4
Genome_build: mm9
Supplementary_files_format_and_content: The processed bigwig files were generated using deeptools(v1.5.11) by normalizing to 1x coverage for the genome size 2150570000

extract_protocol:
Cells were crosslinked  with formaldehyde, lysed and DNA was fragmented into pieces of average 300bp by sonication. Chromatin was precipitated with a specific antibody (anti-H3K4me2;Millipore, 07-030 or anti-H3K27me3; Millipore, CS200603) and protein A-coupled sepharose beads. After elution from the beads, chromatin was decrosslinked and purified via QIAquick PCR Purification Kit according to the manufacturer's manual.
Library preparation for ChIP analysis was performed with the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (Catalog #E7370) according to the manufacturer’s protocol. No size selection was used and amplification was performed using 10 PCR cycles.


title: Input-Ebf1(-/-)::empty

description: Input DNA of Ebf1(-/-)::empty cells

source_name: Ebf1−/− c-Kit+ fetal liver cells, pmys-Empty-IRES-GFP

value: empty


title: Input-Ebf1(-/-)::EBF1wt

description: Input DNA of Ebf1(-/-)::EBF1wt cells

source_name: Ebf1−/− c-Kit+ fetal liver cells, pmys-EBF1wt-IRES-GFP


title: P. chrysogenum-glucose media replicate flask-C

description:
BP-9
P.chrysogenum_FPKMs.txt


title: DNA methylation-Ebf1(-/-)::empty

description: Bisulfite-Seq profile of Ebf1(-/-)::empty


title: DNA methylation-Ebf1(-/-)::EBF1wt

description: Bisulfite-Seq profile of Ebf1(-/-)::EBF1wt


title: H3K4me2-Ebf1(-/-)::EBF1ΔC

description: H3K4me2 ChIP of Ebf1(-/-)::EBF1ΔC cells


title: H3K27me3-Ebf1(-/-)::EBF1ΔC

description: H3K27me3 ChIP of Ebf1(-/-)::EBF1ΔC cells


title: H3K4me2-Ebf1(-/-)::EBF1wt

description: H3K4me2 ChIP of Ebf1(-/-)::EBF1wt-cells


title: H3K27me3-Ebf1(-/-)::EBF1wt

description: H3K27me3 ChIP of Ebf1(-/-)::EBF1wt cells


title: DHS-Ebf1(-/-)::EBF1ΔC

description: DHS of Ebf1(-/-)::EBF1ΔC-DHS

data_processing:
Basecalling and fastq files generation was performed by using Illumina bcl2fastq 1.8.4 (RTA 1.18.64).
The reads were aligned to the mm9 genome assembly using bowtie2 with default configurations.
Duplicated reads were filtered and blacklisted regions were removed using samtools and Picard.
Peaks were identified using MACS2 version 2.1.0.20150731 , q-value cutoff 0.01 and further filtered for p-value 10-7 and fold change 4.
Genome_build: mm9
Supplementary_files_format_and_content: The processed bigwig files were generated using deeptools(v1.5.11) by normalizing to 1x coverage for the genome size 2150570000.

extract_protocol:
For mapping of DNaseI hypersensitive sites, cells were sorted as GFP+ (vector-transduced) or GFP+CD19+ (EBF1wt- and ΔC-transduced). Nuclei were prepared and treated with DNaseI. Subsequently, nuclei were lysed and genomic DNA was extracted using phenol-chloroform-isoamyl alcohol. DNA was concentrated to 250μl and fractionated using 9% sucrose cushion. All fractions of the samples that contain DNA fragments between 100 and 600bp were pooled and purified with Qiagen MinElute Gel extraction Kit.
Library preparation for DHS and ChIP analysis was performed with the NEBNext Ultra DNA Library Prep Kit  according to the manufacturer’s protocol. No size selection was used and amplification was performed using 10 PCR cycles.


title: DHS-Ebf1(-/-)::empty

description: DHS of Ebf1(-/-)::empty

value: empty vector


title: H3K27me3-Ebf1(-/-)::empty

description: H3K27me3 ChIP of Ebf1(-/-)::empty cells


title: H3K4me2-Ebf1(-/-)::empty

description: H3K4me2 ChIP of Ebf1(-/-)::empty cells


title: EBF1ΔC-ChIP

description: EBF1 ChIP of Ebf1(-/-)::EBF1ΔC cells

extract_protocol:
Cells were crosslinked  with formaldehyde, lysed and DNA was fragmented into pieces of average 300bp by sonication. Chromatin was precipitated with a specific antibody (anti-H3K4me2; Millipore, 07-030 or anti-H3K27me3; Millipore, CS200603) and protein A-coupled sepharose beads. After elution from the beads, chromatin was decrosslinked and purified via QIAquick PCR Purification Kit according to the manufacturer's manual.
Library preparation for ChIP analysis was performed with the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (Catalog #E7370) according to the manufacturer’s protocol. No size selection was used and amplification was performed using 10 PCR cycles.


title: ProB-Input-Rep2

description: Input for pro-B EBF1ChIP – Replicate 2

data_processing:
Basecalling and fastq files generation was performed by using Illumina bcl2fastq 1.8.4 (RTA 1.18.64)
The reads were aligned to the mm9 genome assembly using bowtie2 with default configurations
Duplicated reads were filtered and blacklisted regions were removed using samtools and Picard.
Peaks were identified using MACS2 (v2.1.0.20150731) with q-value cutoff 0.01 and further filtered for p-value 10^-10 and fold change 4
Genome_build: mm9
Supplementary_files_format_and_content: The processed bigwig files were generated using deeptools (v1.5.11) by normalizing to 1x coverage for the genome size 2150570000 and a binsize of 50

source_name: Rag2(-/-)  pro-B cells

growth_protocol: Rag2(-/-) pro-B cells were derived from mouse fetal liver and cultured on OP9 cells for short term

extract_protocol:
Cells were crosslinked  with formaldehyde, lysed and DNA was fragmented to an average size of 300bp by sonication. Chromatin was precipitated with a specific antibody (EBF1, produced "in house") and protein A-coupled sepharose beads. After elution from the beads, chromatin was decrosslinked and purified via QIAquick PCR Purification Kit according to the manufacturer's manual.
Library preparation for ChIP analysis was performed with the NEBNext Ultra DNA Library Prep Kit  according to the manufacturer’s protocol. No size selection was used and amplification was performed for 10 PCR cycles.

value: pro-B cells


title: DHS-Ebf1(-/-)::EBF1wt

description: DHS of Ebf1(-/-)::EBF1wt-DHS


title: Nitrogen limited condition, stationary phase 2

city: Berkeley

first: Hiroshi

last: Otani

state: CA

address: 1 Cyclotron Road

department: DOE Joint Genome Institute

institute: Lawerence Berkeley National Laboratory

data_processing:
Sequence Control Software v2.9 was used for basecall
Sequenced reads were trimmed for adaptor sequence
Single-ended sequence reads were scanned 3’ to 5’. Those with a Phred quality value below 20 were trimmed using the FASTQ Quality Trimmer tool of the FASTX-toolkit v 0.0.13.2. Reads of less than 35 bases after trimming were discarded from further analysis.
Trimmed reads were aligned to the R. jostii RHA1 genome using Bowtie 2 v 2.2.3 in end-to-end mode with the “very sensitive” option. Reads of rDNA sequences were omitted in subsequent analyses.
Mapped read counts per gene were obtained using the HTSeq package v 0.5.4p1.
Differential expression (>2-fold change with a false discovery rate-adjusted p value < 0.05) was analyzed using the DESeq2 package v 1.6.3.
Genome_build: ASM1456v1
Supplementary_files_format_and_content: Tab-delimited text files include the number of reads overlapping each feature

organism: Rhodococcus jostii

source_name: Nitrogen limited_stationary phase

growth_protocol: RHA1 strain was cultivated at 30°C under aerobic conditions in M9 minimal medium supplemented with trace elements, thiamin and 20 mM benzoate as growth substrate. For nitrogen-limiting conditions, the concentration of ammonium chloride in the M9 medium was reduced from 1 g l−1 to 0.05 g l−1.

extract_protocol:
Total RNA was extracted using TRIzol® Reagent (Invitrogen) according to the manufacturer’s instructions. The RNA was treated with TURBOTM DNase (Invitrogen) and extracted with phenol-chloroform. The quality and concentration of RNA were assessed using a 2100 Bioanalyzer and the RNA 6000 Nano kit (Agilent). Three µg of total RNA was heated at 65 °C for 5 min, quickly chilled on ice and then mixed with Streptavidin magnetic beads (New England Biolabs) with an equimolar mixture of biotin-labelled probes specific for RHA1 rRNA (23S: AGGAATTTCGCTACCTTAGGATGGTTATAGTTACC and CTTTTC- ACCTTTCCCTCACGGTACTAGTCCGCTAT; 16S: ATATGTCAAACCCAGGTAAGGT- TCTTC) to deplete rRNA. The supernatant was collected and subjected to further rRNA-depletion using the Ribominus™ Transcriptome Isolation Kit (Invitrogen).
One hundred ng of the rRNA-depleted RNA was used for library construction using NEBNext® Ultra Directional RNA Library Prep Kit for Illumina® and NEBNext® Multiplex Oligos for Illumina® (New England Biolabs) according to the manufacturer’s instructions. The samples were analyzed using a Genome Analyzer IIx (Illumina®).

tag: growth phase

value: Stationary

tag: nitrogen

value: Limited

tag: molecule subtype

value: rRNA-depleted RNA


title: Nitrogen excess condition, stationary phase 2

source_name: Nitrogen excess_stationary phase

value: Excess


title: Nitrogen limited condition, stationary phase 1


title: ProB-Input-Rep1

description: Input for pro-B EBF1ChIP – Replicate 1


title: Suz12 Ezh2 fl/fl 6

city: London

first: Richard

last: Jenner

address: 72 Huntley Street

department: Cancer Biology

institute: UCL Cancer Institute

description:
RNA attached to Suz12 in Ezh2 wt cells. Replicate6
Protein crosslinked RNA
Processed data file: Suz12_Ezh2flfl.ensembl59.bed.gz

data_processing:
Fastq files were demultiplexed and aligned using the iCount server
Reads were filtered to remove any peaks within 50 nt of a repeatMasker-annotated repeat or a short non-coding RNA
High-confidence peaks were identified using the 'peaks' option on the iCount server, with the parameters regionAsOne, flank 50, random perms 100 and fdr 0.05
Genome_build: mm9
Supplementary_files_format_and_content: BED files containing number of reads in each Ensembl59 gene for that iCLIP in the score column (column 5)

source_name: mESC

growth_protocol: Cells were cultured on 0.1% gelatin-coated surfaces with KO-DMEM medium, 10% FCS, 5% knockout serum replacement, nonessential amino acids, L-glutamine, 2-mercaptoethanol, antibiotics (Invitrogen), and 1000 U/ml of leukaemia inhibitory factor (Abcam).

extract_protocol:
Individual-nucleotide resolution UV crosslinking and immunoprecipitation protocols were based in protocols available at Huppertz et al. Cells were irradiated with 0.2 J/cm2 of UV light in a Stratalinker 2400 (Stratagene) with bulbs emitting at 254nm, and 200 x10^6 cells per IP were lysed in 1ml of Lysis buffer (50 mM Tris–HCl, pH 7.4; 100 mM NaCl; 1% Igepal CA-630; 0.1% SDS; 0.5% sodium deoxycholate; plus protease inhibitors). Lysates were syringed with a 27G needle and incubated in a Thermomixer at 37°C and 1100 rpm for three minutes after addition of 200U/ml of DNase Turbo and of 20 U/ml of RNase I. IP was set up using 10 μl of α-SUZ12 (Cell Signaling #3737)  as described in iCLIP protocol. Beads were washed 3x with 900 μl high-salt buffer (50 mM Tris-HCl, pH 7.4; 1 M NaCl; 1 mM EDTA; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate and Urea 1M), 2x with 900 μl wash buffer (20 mM Tris-HCl, pH 7.4; 10 mM MgCl2; 0.2% Tween-20) resuspended in 20 μl PNK mix (15 μl water; 4 μl 5x PNK pH 6.5 buffer [350mM Tris-HCl, pH 6.5; 50m MgCl2 25mM dithiothreitol]; 0.5 μl PNK enzyme; 0.5 μl RNasin [Promega]) and incubated 30 min at 37°C 1100 rpm. Beads were washed 3x with 900 μl high-salt buffer, 2x with 900 μl wash buffer, resuspended in beads in 20 μl ligation mix (9 μl water; 4 μl 4x ligation buffer [200 mM Tris-HCl; 40m MM gCl2; 40 mM dithiothreitol]; 1 μl RNA ligase [NEB]; 0.5 μl RNasin [Promega]; 1.5 μl pre-adenylated linker L3 [20 μM]; 4 μl PEG400 [81170, Sigma]), ligation was performed over night at 16°C and 1100 rpm. Beads were washed 3x with high-salt buffer, 2x wash buffer and 20% of the beads were incubated with 8 μl of hot PNK mix (0.4 μl PNK [NEB]; 0.8 μl 32P-γ-ATP; 0.8 μl 10x PNK buffer [NEB]; 6 μl water), radiolabelled and non-radiolabelled beads were pooled again and resuspended in 20 μl in Nupage loading buffer with reducing agent and run on a 4-12% NuPAGE Bis-Tris gel (Invitrogen) in MOPS buffer according to the manufacturer's instructions. Protein-RNA complexes were transferred to a nitrocellulose membrane (Hybond, GE Healthcare) washed 2x with PBS1x and exposed to a Fuji film. Using the auto-radiograph as a mask RNA-protein complexes attached to the membrane were isolated and treated with 200 μl PK buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA) and 10 μl proteinase K (Roche, 03115828001) for 20 minutes at 1,100 rpm and 37°C. 200 μl of PK-urea buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA; 7 M urea) was added and a second incubation was performed for 20 minutes at 37°C and 1100 rpm. Solution was collected and RNA was isolated by phenol: chloroform extraction and ethanol precipitation over night at -20°C.
RNA pellet was resuspended in 7.25 μl RNA/primer mix (6.25 μl water; 0.5 μl Rclip primer [0.5 uM]; 0.5 μl dNTP mix [10mM]) and transferred in a 0.2 ml tube and incubated at 70°C. Retrotranscription was performed by addition of 2 μl 5x RT buffer; 0.5 μl 0.1M DTT; 0.25 μl Superscript III reverse transcriptase [Invitrogen] and incubation 20 min at 42°C, 40 min at 50°C and 5 min at 80°C. cDNA was fractionated running samples on a precast 6% TBE-urea gel 180V 40 min and isolating bands at 250-180 nt (top), 120-180 nt (high), 85-120 nt (medium) and 70-85 nt (low). Gel slices were submerged and crushed in TE buffer, incubated 1h at 37°C 1100 rpm, flash frozen, and a second incubation was performed 1h at 37°C 1100 rpm. cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C. cDNA was resuspended in 8 μl ligation mix (6.5 μl water; 0.8 μl 10x CircLigase Buffer II; 0.4 μl 50 mM MnCl2; 0.3 μl Circligase II [Epicentre]) transferred into a 0.2 tube and incubated for 1 h at 60°C for circularization. Samples were incubated 30 μl oligo annealing mix (26 μl water; 3 μl FastDigest Buffer [Fermentas]; 1 μl cut_oligo [10 μM]), oligo was annealed by temperature gradient and circular cDNA was re-linearized incubating samples with 2 μl BamHI (Fast Fermentas).
cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C and pellet were resuspended in 20 μl of H20. Amplification was performed using 10 μl cDNA; 1 μl primer mix P5/P3 solexa, 10 μM each; 20 μl Accuprime Supermix 1 enzyme [Invitrogen], 9 μl H20 and the following PCR programme: 94°C for 2 min, [94°C for 15 sec, 65°C for 30 sec, 68°C for 30 sec]n cycles, 68°C for 3 min, 4°C, with a number of cycles optimized previously. Library integrity was analysed using 8 μl PCR product with 8 μl of 5x TBE loading buffer and loading on a precast 6% TBE gel (Invitrogen). Gel was stained with Sybrgreen I (Invitrogen) and analyse with a gel imager. Library concentration was determined using the KAPA Universal Library Quantification kit, according to the manufacturer’s instructions and library concentration was corrected by multiplication by 0.38 to improve cluster density. Libraries were sequenced using Illumina HiSeq 2500 sequencers (50 bp single-end reads).
RNA pellet was resuspended in 7.25 μl RNA/primer mix (6.25 μl water; 0.5 μl Rclip primer [0.5 uM]; 0.5 μl dNTP mix [10mM]) and transferred in a 0.2 mL tube and incubated at 70°C. Retrotranscription was performed by addition of 2 μl 5x RT buffer; 0.5 μl 0.1M DTT; 0.25 μl Superscript III reverse transcriptase [Invitrogen] and incubation 20 min at 42°C, 40 min at 50°C and 5 min at 80°C. cDNA was fractionated running samples on a precast 6% TBE-urea gel 180V 40 min and isolating bands at 250-180 nt (top), 120-180 nt (high), 85-120 nt (medium) and 70-85 nt (low). Gel slices were submerged and crushed in TE buffer, incubated 1h at 37°C 1100 rpm, flash frozen, and a second incubation was performed 1h at 37°C 1100 rpm. cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C. cDNA was resuspended in 8 μl ligation mix (6.5 μl water; 0.8 μl 10x CircLigase Buffer II; 0.4 μl 50 mM MnCl2; 0.3 μl; Circligase II [Epicentre]) transferred into a 0.2 tube and incubated for 1 h at 60°C for circularization. Samples were incubated 30 μl oligo annealing mix (26 μl water; 3 μl FastDigest Buffer [Fermentas]; 1 μl cut_oligo [10 μM]), oligo was annealed by temperature gradient and circular cDNA was re-linearized incubating samples with 2 μl BamHI (Fast Fermentas).
cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C and pellet were resuspended in 20 μl of H20. Amplification was performed using 10 μl cDNA; 1 μl primer mix P5/P3 solexa, 10 μM each; 20 μl Accuprime Supermix 1 enzyme [Invitrogen], 9H20 and the following PCR programme: 94°C for 2 min, [94°C for 15 sec, 65°C for 30 sec, 68°C for 30 sec]n cycles, 68°C for 3 min, 4°C, with a number of cycles optimized previously. Library integrity was analysed using 8 μl PCR product with 8 μl of 5x TBE loading buffer and loading on a precast 6% TBE gel (Invitrogen). Gel was stained with Sybrgreen I (Invitrogen) and analyse with a gel imager. Concentration was determined using KAPPA kit according manufacturer instructions.

treatment_protocol: Ezh2fl/fl mESC were treated with 4-hydroxy-tamoxifen for 96 hours (800 nM, Sigma) when indicated to generate Ezh2∆/∆ cells

value: m ESC Ezh2 fl/fl

value: No Tamoxifen

value: Ezh2 wild type

tag: ip antibody

value: Suz12 Cell signalling #3737


title: Eed

description:
RNA attached to Eed in Ezh2 wt cells.
Protein crosslinked RNA
Processed data file: Eed.ensembl59.bed.gz

extract_protocol:
Individual-nucleotide resolution UV crosslinking and immunoprecipitation protocols were based in protocols available at Huppertz et al. Cells were irradiated with 0.15 J/cm2 of UV light in a Stratalinker 2400 (Stratagene) with bulbs emitting at 254nm, and 200 x10^6 cells per IP were lysed in 1ml of Lysis buffer (50 mM Tris–HCl, pH 7.4; 100 mM NaCl; 1% Igepal CA-630; 0.1% SDS; 0.5% sodium deoxycholate; plus protease inhibitors). Lysates were syringed with a 27G needle and incubated in a Thermomixer at 37°C and 1100 rpm for three minutes after addition of 200U/ml of DNase Turbo and of 20 U/ml of RNase I. IP was set up using 10 μg of α-Myc (Cell Signaling, #2276)  as described in iCLIP protocol. Beads were washed 3x with 900 μl high-salt buffer (50 mM Tris-HCl, pH 7.4; 1 M NaCl; 1 mM EDTA; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate and Urea 1M), 2x with 900 μl wash buffer (20 mM Tris-HCl, pH 7.4; 10 mM MgCl2; 0.2% Tween-20) resuspended in 20 μl PNK mix (15 μl water; 4 μl 5x PNK pH 6.5 buffer [350mM Tris-HCl, pH 6.5; 50m MgCl2 25mM dithiothreitol]; 0.5 μl PNK enzyme; 0.5 μl RNasin [Promega]) and incubated 30 min at 37°C 1100 rpm. Beads were washed 3x with 900 μl high-salt buffer, 2x with 900 μl wash buffer, resuspended in beads in 20 μl ligation mix (9 μl water; 4 μl 4x ligation buffer [200 mM Tris-HCl; 40m MM gCl2; 40 mM dithiothreitol]; 1 μl RNA ligase [NEB]; 0.5 μl RNasin [Promega]; 1.5 μl pre-adenylated linker L3 [20 μM]; 4 μl PEG400 [81170, Sigma]), ligation was performed over night at 16°C and 1100 rpm. Beads were washed 3x with high-salt buffer, 2x wash buffer and 20% of the beads were incubated with 8 μl of hot PNK mix (0.4 μl PNK [NEB]; 0.8 μl 32P-γ-ATP; 0.8 μl 10x PNK buffer [NEB]; 6 μl water), radiolabelled and non-radiolabelled beads were pooled again and resuspended in 20 μl in Nupage loading buffer with reducing agent and run on a 4-12% NuPAGE Bis-Tris gel (Invitrogen) in MOPS buffer according to the manufacturer's instructions. Protein-RNA complexes were transferred to a nitrocellulose membrane (Hybond, GE Healthcare) washed 2x with PBS1x and exposed to a Fuji film. Using the auto-radiograph as a mask RNA-protein complexes attached to the membrane were isolated and treated with 200 μl PK buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA) and 10 μl proteinase K (Roche, 03115828001) for 20 minutes at 1,100 rpm and 37°C. 200 μl of PK-urea buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA; 7 M urea) was added and a second incubation was performed for 20 minutes at 37°C and 1100 rpm. Solution was collected and RNA was isolated by phenol: chloroform extraction and ethanol precipitation over night at -20°C.
RNA pellet was resuspended in 7.25 μl RNA/primer mix (6.25 μl water; 0.5 μl Rclip primer [0.5 uM]; 0.5 μl dNTP mix [10mM]) and transferred in a 0.2 ml tube and incubated at 70°C. Retrotranscription was performed by addition of 2 μl 5x RT buffer; 0.5 μl 0.1M DTT; 0.25 μl Superscript III reverse transcriptase [Invitrogen] and incubation 20 min at 42°C, 40 min at 50°C and 5 min at 80°C. cDNA was fractionated running samples on a precast 6% TBE-urea gel 180V 40 min and isolating bands at 250-180 nt (top), 120-180 nt (high), 85-120 nt (medium) and 70-85 nt (low). Gel slices were submerged and crushed in TE buffer, incubated 1h at 37°C 1100 rpm, flash frozen, and a second incubation was performed 1h at 37°C 1100 rpm. cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C. cDNA was resuspended in 8 μl ligation mix (6.5 μl water; 0.8 μl 10x CircLigase Buffer II; 0.4 μl 50 mM MnCl2; 0.3 μl Circligase II [Epicentre]) transferred into a 0.2 tube and incubated for 1 h at 60°C for circularization. Samples were incubated 30 μl oligo annealing mix (26 μl water; 3 μl FastDigest Buffer [Fermentas]; 1 μl cut_oligo [10 μM]), oligo was annealed by temperature gradient and circular cDNA was re-linearized incubating samples with 2 μl BamHI (Fast Fermentas).
cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C and pellet were resuspended in 20 μl of H20. Amplification was performed using 10 μl cDNA; 1 μl primer mix P5/P3 solexa, 10 μM each; 20 μl Accuprime Supermix 1 enzyme [Invitrogen], 9 μl H20 and the following PCR programme: 94°C for 2 min, [94°C for 15 sec, 65°C for 30 sec, 68°C for 30 sec]n cycles, 68°C for 3 min, 4°C, with a number of cycles optimized previously. Library integrity was analysed using 8 μl PCR product with 8 μl of 5x TBE loading buffer and loading on a precast 6% TBE gel (Invitrogen). Gel was stained with Sybrgreen I (Invitrogen) and analyse with a gel imager. Library concentration was determined using the KAPA Universal Library Quantification kit, according to the manufacturer’s instructions and library concentration was corrected by multiplication by 0.38 to improve cluster density. Libraries were sequenced using Illumina HiSeq 2500 sequencers (50 bp single-end reads).

value: m ESC cKO Eed 4d2

value: α-Myc  Cell Signaling, #2276


title: Nitrogen limited condition, exponential phase 1

source_name: Nitrogen limited_exponential phase

value: Expenential


title: Nitrogen excess condition, stationary phase 1


title: Suz12 Jarid2 WT

description:
RNA attached to Suz12 in Jarid2 WT cells
Protein crosslinked RNA
Processed data file: Suz12_Jarid2WT.ensembl59.bed.gz

extract_protocol:
Individual-nucleotide resolution UV crosslinking and immunoprecipitation protocols were based in protocols available at Huppertz et al. Cells were irradiated with 0.15 J/cm2 of UV light in a Stratalinker 2400 (Stratagene) with bulbs emitting at 254nm, and 200 x10^6 cells per IP were lysed in 1ml of Lysis buffer (50 mM Tris–HCl, pH 7.4; 100 mM NaCl; 1% Igepal CA-630; 0.1% SDS; 0.5% sodium deoxycholate; plus protease inhibitors). Lysates were syringed with a 27G needle and incubated in a Thermomixer at 37°C and 1100 rpm for three minutes after addition of 200U/ml of DNase Turbo and of 20 U/ml of RNase I. IP was set up using 10 μl of α-SUZ12 (Cell Signaling #3737)  as described in iCLIP protocol. Beads were washed 3x with 900 μl high-salt buffer (50 mM Tris-HCl, pH 7.4; 1 M NaCl; 1 mM EDTA; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate and Urea 1M), 2x with 900 μl wash buffer (20 mM Tris-HCl, pH 7.4; 10 mM MgCl2; 0.2% Tween-20) resuspended in 20 μl PNK mix (15 μl water; 4 μl 5x PNK pH 6.5 buffer [350mM Tris-HCl, pH 6.5; 50m MgCl2 25mM dithiothreitol]; 0.5 μl PNK enzyme; 0.5 μl RNasin [Promega]) and incubated 30 min at 37°C 1100 rpm. Beads were washed 3x with 900 μl high-salt buffer, 2x with 900 μl wash buffer, resuspended in beads in 20 μl ligation mix (9 μl water; 4 μl 4x ligation buffer [200 mM Tris-HCl; 40m MM gCl2; 40 mM dithiothreitol]; 1 μl RNA ligase [NEB]; 0.5 μl RNasin [Promega]; 1.5 μl pre-adenylated linker L3 [20 μM]; 4 μl PEG400 [81170, Sigma]), ligation was performed over night at 16°C and 1100 rpm. Beads were washed 3x with high-salt buffer, 2x wash buffer and 20% of the beads were incubated with 8 μl of hot PNK mix (0.4 μl PNK [NEB]; 0.8 μl 32P-γ-ATP; 0.8 μl 10x PNK buffer [NEB]; 6 μl water), radiolabelled and non-radiolabelled beads were pooled again and resuspended in 20 μl in Nupage loading buffer with reducing agent and run on a 4-12% NuPAGE Bis-Tris gel (Invitrogen) in MOPS buffer according to the manufacturer's instructions. Protein-RNA complexes were transferred to a nitrocellulose membrane (Hybond, GE Healthcare) washed 2x with PBS1x and exposed to a Fuji film. Using the auto-radiograph as a mask RNA-protein complexes attached to the membrane were isolated and treated with 200 μl PK buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA) and 10 μl proteinase K (Roche, 03115828001) for 20 minutes at 1,100 rpm and 37°C. 200 μl of PK-urea buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA; 7 M urea) was added and a second incubation was performed for 20 minutes at 37°C and 1100 rpm. Solution was collected and RNA was isolated by phenol: chloroform extraction and ethanol precipitation over night at -20°C.
RNA pellet was resuspended in 7.25 μl RNA/primer mix (6.25 μl water; 0.5 μl Rclip primer [0.5 uM]; 0.5 μl dNTP mix [10mM]) and transferred in a 0.2 ml tube and incubated at 70°C. Retrotranscription was performed by addition of 2 μl 5x RT buffer; 0.5 μl 0.1M DTT; 0.25 μl Superscript III reverse transcriptase [Invitrogen] and incubation 20 min at 42°C, 40 min at 50°C and 5 min at 80°C. cDNA was fractionated running samples on a precast 6% TBE-urea gel 180V 40 min and isolating bands at 250-180 nt (top), 120-180 nt (high), 85-120 nt (medium) and 70-85 nt (low). Gel slices were submerged and crushed in TE buffer, incubated 1h at 37°C 1100 rpm, flash frozen, and a second incubation was performed 1h at 37°C 1100 rpm. cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C. cDNA was resuspended in 8 μl ligation mix (6.5 μl water; 0.8 μl 10x CircLigase Buffer II; 0.4 μl 50 mM MnCl2; 0.3 μl Circligase II [Epicentre]) transferred into a 0.2 tube and incubated for 1 h at 60°C for circularization. Samples were incubated 30 μl oligo annealing mix (26 μl water; 3 μl FastDigest Buffer [Fermentas]; 1 μl cut_oligo [10 μM]), oligo was annealed by temperature gradient and circular cDNA was re-linearized incubating samples with 2 μl BamHI (Fast Fermentas).
cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C and pellet were resuspended in 20 μl of H20. Amplification was performed using 10 μl cDNA; 1 μl primer mix P5/P3 solexa, 10 μM each; 20 μl Accuprime Supermix 1 enzyme [Invitrogen], 9 μl H20 and the following PCR programme: 94°C for 2 min, [94°C for 15 sec, 65°C for 30 sec, 68°C for 30 sec]n cycles, 68°C for 3 min, 4°C, with a number of cycles optimized previously. Library integrity was analysed using 8 μl PCR product with 8 μl of 5x TBE loading buffer and loading on a precast 6% TBE gel (Invitrogen). Gel was stained with Sybrgreen I (Invitrogen) and analyse with a gel imager. Library concentration was determined using the KAPA Universal Library Quantification kit, according to the manufacturer’s instructions and library concentration was corrected by multiplication by 0.38 to improve cluster density. Libraries were sequenced using Illumina HiSeq 2500 sequencers (50 bp single-end reads).

value: Plus Tamoxifen

value: Jarid2 WT


title: Nitrogen excess condition, exponential phase 2

source_name: Nitrogen excess_exponential phase


title: Suz12 Suz12KO 2

description:
RNA immunoprecipitated by an anti-Suz12 antibody in Suz12 KO cells
Protein crosslinked RNA
Processed data file: Suz12_Suz12KO.ensembl59.bed.gz

extract_protocol:
Individual-nucleotide resolution UV crosslinking and immunoprecipitation protocols were based in protocols available at Huppertz et al. Cells were irradiated with 0.2 J/cm2 of UV light in a Stratalinker 2400 (Stratagene) with bulbs emitting at 254nm, and 200 x10^6 cells per IP were lysed in 1ml of Lysis buffer (50 mM Tris–HCl, pH 7.4; 100 mM NaCl; 1% Igepal CA-630; 0.1% SDS; 0.5% sodium deoxycholate; plus protease inhibitors). Lysates were syringed with a 27G needle and incubated in a Thermomixer at 37°C and 1100 rpm for three minutes after addition of 200U/ml of DNase Turbo and of 20 U/ml of RNase I. IP was set up using 10 μl of α-SUZ12 (Cell Signaling #3737)  as described in iCLIP protocol. Beads were washed 3x with 900 μl high-salt buffer (50 mM Tris-HCl, pH 7.4; 1 M NaCl; 1 mM EDTA; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate and Urea 1M), 2x with 900 μl wash buffer (20 mM Tris-HCl, pH 7.4; 10 mM MgCl2; 0.2% Tween-20) resuspended in 20 μl PNK mix (15 μl water; 4 μl 5x PNK pH 6.5 buffer [350mM Tris-HCl, pH 6.5; 50m MgCl2 25mM dithiothreitol]; 0.5 μl PNK enzyme; 0.5 μl RNasin [Promega]) and incubated 30 min at 37°C 1100 rpm. Beads were washed 3x with 900 μl high-salt buffer, 2x with 900 μl wash buffer, resuspended in beads in 20 μl ligation mix (9 μl water; 4 μl 4x ligation buffer [200 mM Tris-HCl; 40m MM gCl2; 40 mM dithiothreitol]; 1 μl RNA ligase [NEB]; 0.5 μl RNasin [Promega]; 1.5 μl pre-adenylated linker L3 [20 μM]; 4 μl PEG400 [81170, Sigma]), ligation was performed over night at 16°C and 1100 rpm. Beads were washed 3x with high-salt buffer, 2x wash buffer and 20% of the beads were incubated with 8 μl of hot PNK mix (0.4 μl PNK [NEB]; 0.8 μl 32P-γ-ATP; 0.8 μl 10x PNK buffer [NEB]; 6 μl water), radiolabelled and non-radiolabelled beads were pooled again and resuspended in 20 μl in Nupage loading buffer with reducing agent and run on a 4-12% NuPAGE Bis-Tris gel (Invitrogen) in MOPS buffer according to the manufacturer's instructions. Protein-RNA complexes were transferred to a nitrocellulose membrane (Hybond, GE Healthcare) washed 2x with PBS1x and exposed to a Fuji film. Using the auto-radiograph as a mask RNA-protein complexes attached to the membrane were isolated and treated with 200 μl PK buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA) and 10 μl proteinase K (Roche, 03115828001) for 20 minutes at 1,100 rpm and 37°C. 200 μl of PK-urea buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA; 7 M urea) was added and a second incubation was performed for 20 minutes at 37°C and 1100 rpm. Solution was collected and RNA was isolated by phenol: chloroform extraction and ethanol precipitation over night at -20°C.
RNA pellet was resuspended in 7.25 μl RNA/primer mix (6.25 μl water; 0.5 μl Rclip primer [0.5 uM]; 0.5 μl dNTP mix [10mM]) and transferred in a 0.2 ml tube and incubated at 70°C. Retrotranscription was performed by addition of 2 μl 5x RT buffer; 0.5 μl 0.1M DTT; 0.25 μl Superscript III reverse transcriptase [Invitrogen] and incubation 20 min at 42°C, 40 min at 50°C and 5 min at 80°C. cDNA was fractionated running samples on a precast 6% TBE-urea gel 180V 40 min and isolating bands at 250-180 nt (top), 120-180 nt (high), 85-120 nt (medium) and 70-85 nt (low). Gel slices were submerged and crushed in TE buffer, incubated 1h at 37°C 1100 rpm, flash frozen, and a second incubation was performed 1h at 37°C 1100 rpm. cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C. cDNA was resuspended in 8 μl ligation mix (6.5 μl water; 0.8 μl 10x CircLigase Buffer II; 0.4 μl 50 mM MnCl2; 0.3 μl Circligase II [Epicentre]) transferred into a 0.2 tube and incubated for 1 h at 60°C for circularization. Samples were incubated 30 μl oligo annealing mix (26 μl water; 3 μl FastDigest Buffer [Fermentas]; 1 μl cut_oligo [10 μM]), oligo was annealed by temperature gradient and circular cDNA was re-linearized incubating samples with 2 μl BamHI (Fast Fermentas).
cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C and pellet were resuspended in 20 μl of H20. Amplification was performed using 10 μl cDNA; 1 μl primer mix P5/P3 solexa, 10 μM each; 20 μl Accuprime Supermix 1 enzyme [Invitrogen], 9 μl H20 and the following PCR programme: 94°C for 2 min, [94°C for 15 sec, 65°C for 30 sec, 68°C for 30 sec]n cycles, 68°C for 3 min, 4°C, with a number of cycles optimized previously. Library integrity was analysed using 8 μl PCR product with 8 μl of 5x TBE loading buffer and loading on a precast 6% TBE gel (Invitrogen). Gel was stained with Sybrgreen I (Invitrogen) and analyse with a gel imager. Library concentration was determined using the KAPA Universal Library Quantification kit, according to the manufacturer’s instructions and library concentration was corrected by multiplication by 0.38 to improve cluster density. Libraries were sequenced using Illumina HiSeq 2500 sequencers (50 bp single-end reads).

value: m ESC Suz12 -/- (from Pasini lab)

value: Suz12 KO


title: Nitrogen excess condition, exponential phase 1


title: p300_minusRNase

description: DNA attached to p300 after mock treatment

data_processing:
Adaptor sequences were removed using fastq-mcf -t 0.0001
Low quality bases were removed using seqtk trimfq -q 0.01
Reads were aligned to mm9 using bowtie2 with the parameters --very-sensitive -p 4
BigWig files were generated using bedGraphToBigWig
Genome_build: mm9
Supplementary_files_format_and_content: bigWig

extract_protocol:
Lysates were clarified from sonicated nuclei using a Diagenode Bioruptor (3 rounds of 12 cycles) and histone-DNA complexes were isolated with antibody.
Libraries were prepared according to NEB instructions accompanying the NEBNext® DNA Library Prep Kit for Illumina. 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 10-15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel.

tag: treatment/genotype

value: No Rnase

value: Bethyl A300-358A


title: Suz12_plusRNase 2

description: DNA attached to Suz12 after RNase treatment

value: Rnase

value: Suz12 Abcam ab12073


title: p300_plusRNase

description: DNA attached to p300 after RNase treatment


title: Suz12 Suz12KO 1


title: P. falciparum Dd2_w/CQ

city: Pondicherry

first: Hiasindh Ashmi

last: Antony

state: Pondicherry

address: Dhanvantri Nagar

department: Microbiology

country: India

institute: Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER)

description: Grown in the presence of chloroquine using in vitro method

data_processing:
Quality check and Base trimming: FASTQC report is generated using FASTQC program. Base trimming is done using in house perl program
Adapter trimming: Using Cutadapt program
Contamination Removal: From the Trimmed paired-end reads, we removed unwanted sequences, and especially non-polyA tailed RNAs. The unwanted ribosomal RNAs, transfer RNAs, adapter sequences and others. The contamination removal step had been performed using the help of bowtie2 (version 2.1.0), in-house Perl scripts and picard tools(version 1.85)
Read alignment: The alignment was performed using Tophat program (version 2.0.8) with default parameters.
Expression estimation and differential expression analysis: After aligning the reads with reference gene model, the aligned reads were used for estimating expression of the genes and transcripts; using cufflinks program (version= 2.2.0). Differential expression analysis was performed using cuffdiff program
Genome_build: ftp://ftp.ensemblgenomes.org/pub/release-26/protists/fasta/plasmodium_falciparum/dna/Plasmodium_falciparum.ASM276v1.26.dna.toplevel.fa.gz
Supplementary_files_format_and_content: CuffDiff output

organism: Plasmodium falciparum

source_name: Infected red blood cells

growth_protocol: P. falciparum strains (3D7 and Dd2) were maintained in continuous culture by in vitro method, in ‘O’ positive red blood cells at 1-5 % parasitemia and 5 % haematocrit in RPMI 1640 (Gibco) supplemented with 25 mM HEPES buffer, 2.1 g/L sodium bicarbonate, 200 µg/ml gentamycin, 5 % albumax (Gibco) and 0.05 % hypoxanthine (Sigma). The resistant strain Dd2 was also grown in the presence of chloroquine with MIC concentration,160 nmol/l, with the same growth condition mentioned above. The parasite cells were harvested at 48 hr havving ~ 5 % parasitemia with 90 % trophozoites.

extract_protocol:
Total RNA was extracted from the infected red blood cells using Trizol method and mRNA purification was done by magnetic bead method.
cDNA synthesis was performed using reverse transcriptase and random primer and double strand cDNA was purified by AMPure XP beads. End repair was carried out to convert the overhangs into blunt ends and 3' adenylation prevent ligation to one another during adapter ligation reaction. Adaptor ligation was performed to ligate multiple indexing adapters to the ends of ds cDNA and helps in the hybridization onto flow cell. cDNA fragments were enriched using PCR which selectively enrich fragments having adapter on both ends and amplify amount of DNA in library.

tag: phenotype

value: Chloroquine resistant

tag: cell stage

value: 90 % trophozoite at 48 hr post infection


title: P. falciparum Dd2

description: Grown in the absence of chloroquine using in vitro method


title: chp2D_Rep_1

city: Memphis

last: Finkelstein

state: TN

address: 332 N. Lauderdale St.

department: Computational Biology

institute: St Jude Children's Research Hospital

data_processing:
Illumina Casava1.8.2 software used for basecalling.
Reads were mapped to Schizosaccharomyces pombe using GSNAP (version 2013-02-25)
Gene level FPKM was estimated using in house perl script
Genome_build: ftp://ftp.ebi.ac.uk/pub/databases/pombase/pombe/Chromosome_Dumps/Schizosaccharomyces_pombe.ASM294v2.20.dna.genome.fa
Supplementary_files_format_and_content: tab-delimited text files include gene level FPKM valuse for each Sample

organism: Schizosaccharomyces pombe

source_name: Fission yeast

growth_protocol: Fission yeast (25 ml cultures) were grown to 2.5 x10-6 cells/ ml in YES media at 25C at 170 rpm

extract_protocol:
RNA was prepared as described (Creamer et al., MCB 34(11):2046-61, 2014)
Ribosomal RNA was removed using Ribo-Zero Gold rRNA Removal Kit (Yeast) (Illumina, San Diego, CA.) Libraries were prepared from the rRNA depleted RNA using the TruSeq Stranded Total RNA Library Prep Kit with modifications (Brugger et al.)

value: h- chp2D::KanR ade6-210 arg3-D4 his3-D1 leu1-32 ura4-D18


title: P. falciparum 3D7

value: Chloroquine sensitive


title: mit1D_Rep_3

value: h- mit1D::Natmx6 ade6-210 arg3-D4 his3-D1 leu1-32 ura4-D18


title: chp2D_Rep_2


title: chp2D_Rep_3


title: Suz12_minusRNase

description: DNA attached to Suz12 after mock treatment

value: m ESC Ezh2 fl/fl

value: Ezh2 fl/fl wildtype


title: GFP

description:
RNA attached to Suz12 in Jarid2 KO cells
Protein crosslinked RNA
Processed data file: GFP.ensembl59.bed.gz

extract_protocol:
Individual-nucleotide resolution UV crosslinking and immunoprecipitation protocols were based in protocols available at Huppertz et al. Cells were irradiated with 0.2 J/cm2 of UV light in a Stratalinker 2400 (Stratagene) with bulbs emitting at 254nm, and 200 x10^6 cells per IP were lysed in 1ml of Lysis buffer (50 mM Tris–HCl, pH 7.4; 100 mM NaCl; 1% Igepal CA-630; 0.1% SDS; 0.5% sodium deoxycholate; plus protease inhibitors). Lysates were syringed with a 27G needle and incubated in a Thermomixer at 37°C and 1100 rpm for three minutes after addition of 200U/ml of DNase Turbo and of 20 U/ml of RNase I. IP was set up using 10 μg of α-GFP (Abcam ab290)  as described in iCLIP protocol. Beads were washed 3x with 900 μl high-salt buffer (50 mM Tris-HCl, pH 7.4; 1 M NaCl; 1 mM EDTA; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate and Urea 1M), 2x with 900 μl wash buffer (20 mM Tris-HCl, pH 7.4; 10 mM MgCl2; 0.2% Tween-20) resuspended in 20 μl PNK mix (15 μl water; 4 μl 5x PNK pH 6.5 buffer [350mM Tris-HCl, pH 6.5; 50m MgCl2 25mM dithiothreitol]; 0.5 μl PNK enzyme; 0.5 μl RNasin [Promega]) and incubated 30 min at 37°C 1100 rpm. Beads were washed 3x with 900 μl high-salt buffer, 2x with 900 μl wash buffer, resuspended in beads in 20 μl ligation mix (9 μl water; 4 μl 4x ligation buffer [200 mM Tris-HCl; 40m MM gCl2; 40 mM dithiothreitol]; 1 μl RNA ligase [NEB]; 0.5 μl RNasin [Promega]; 1.5 μl pre-adenylated linker L3 [20 μM]; 4 μl PEG400 [81170, Sigma]), ligation was performed over night at 16°C and 1100 rpm. Beads were washed 3x with high-salt buffer, 2x wash buffer and 20% of the beads were incubated with 8 μl of hot PNK mix (0.4 μl PNK [NEB]; 0.8 μl 32P-γ-ATP; 0.8 μl 10x PNK buffer [NEB]; 6 μl water), radiolabelled and non-radiolabelled beads were pooled again and resuspended in 20 μl in Nupage loading buffer with reducing agent and run on a 4-12% NuPAGE Bis-Tris gel (Invitrogen) in MOPS buffer according to the manufacturer's instructions. Protein-RNA complexes were transferred to a nitrocellulose membrane (Hybond, GE Healthcare) washed 2x with PBS1x and exposed to a Fuji film. Using the auto-radiograph as a mask RNA-protein complexes attached to the membrane were isolated and treated with 200 μl PK buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA) and 10 μl proteinase K (Roche, 03115828001) for 20 minutes at 1,100 rpm and 37°C. 200 μl of PK-urea buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA; 7 M urea) was added and a second incubation was performed for 20 minutes at 37°C and 1100 rpm. Solution was collected and RNA was isolated by phenol: chloroform extraction and ethanol precipitation over night at -20°C.
RNA pellet was resuspended in 7.25 μl RNA/primer mix (6.25 μl water; 0.5 μl Rclip primer [0.5 uM]; 0.5 μl dNTP mix [10mM]) and transferred in a 0.2 ml tube and incubated at 70°C. Retrotranscription was performed by addition of 2 μl 5x RT buffer; 0.5 μl 0.1M DTT; 0.25 μl Superscript III reverse transcriptase [Invitrogen] and incubation 20 min at 42°C, 40 min at 50°C and 5 min at 80°C. cDNA was fractionated running samples on a precast 6% TBE-urea gel 180V 40 min and isolating bands at 250-180 nt (top), 120-180 nt (high), 85-120 nt (medium) and 70-85 nt (low). Gel slices were submerged and crushed in TE buffer, incubated 1h at 37°C 1100 rpm, flash frozen, and a second incubation was performed 1h at 37°C 1100 rpm. cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C. cDNA was resuspended in 8 μl ligation mix (6.5 μl water; 0.8 μl 10x CircLigase Buffer II; 0.4 μl 50 mM MnCl2; 0.3 μl Circligase II [Epicentre]) transferred into a 0.2 tube and incubated for 1 h at 60°C for circularization. Samples were incubated 30 μl oligo annealing mix (26 μl water; 3 μl FastDigest Buffer [Fermentas]; 1 μl cut_oligo [10 μM]), oligo was annealed by temperature gradient and circular cDNA was re-linearized incubating samples with 2 μl BamHI (Fast Fermentas).
cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C and pellet were resuspended in 20 μl of H20. Amplification was performed using 10 μl cDNA; 1 μl primer mix P5/P3 solexa, 10 μM each; 20 μl Accuprime Supermix 1 enzyme [Invitrogen], 9 μl H20 and the following PCR programme: 94°C for 2 min, [94°C for 15 sec, 65°C for 30 sec, 68°C for 30 sec]n cycles, 68°C for 3 min, 4°C, with a number of cycles optimized previously. Library integrity was analysed using 8 μl PCR product with 8 μl of 5x TBE loading buffer and loading on a precast 6% TBE gel (Invitrogen). Gel was stained with Sybrgreen I (Invitrogen) and analyse with a gel imager. Library concentration was determined using the KAPA Universal Library Quantification kit, according to the manufacturer’s instructions and library concentration was corrected by multiplication by 0.38 to improve cluster density. Libraries were sequenced using Illumina HiSeq 2500 sequencers (50 bp single-end reads).

value: No Tamoxifen

value: Ezh2 wild type

value: GFP Abcam #ab290


title: mit1D_Rep_2


title: Suz12_minusRNase 2


title: Suz12_plusRNase


title: Nitrogen limited condition, exponential phase 2

value: Expenential

value: Limited

value: rRNA-depleted RNA


title: mit1D_Rep_1


title: Suz12 Ezh2 fl/fl 5

description:
RNA attached to Suz12 in Ezh2 wt cells. Replicate5
Protein crosslinked RNA
Processed data file: Suz12_Ezh2flfl.ensembl59.bed.gz

value: Suz12 Cell signalling #3737


title: clr3D_Rep_2

value: h- clr3D::kanMX6  ade6-210   arg3D  his3D  leu1-32  ura4-D18


title: Ezh2_plusTmx

description: DNA attached to Ezh2 in Ezh2 KO cells.

value: Plus Tamoxifen

value: Ezh2 KO

value: Ezh2 Cell Signaling #3147


title: input_plusTmx

description: DNA  in Ezh2 KO cells.

value: Whole cell extract


title: Suz12_plusTmx

description: DNA attached to Suz12 in Ezh2 KO cells.

value: Suz12 Abcam ab12073


title: Suz12 Ezh2 fl/fl 4

description:
RNA attached to Suz12 in Ezh2 wt cells. Replicate4
Protein crosslinked RNA
Processed data file: Suz12_Ezh2flfl.ensembl59.bed.gz


title: clr3D_Rep_3


title: clr3D_Rep_1


title: input_minusTmx

description: DNA  in Ezh2 wt cells.


title: clr2D_Rep_3

value: h- clr2D::his7+ ade6-216  arg3D  his3D  leu1-32  ura4-D18


title: clr2D_Rep_2


title: Ezh2_minusTmx

description: DNA attached to Ezh2 in Ezh2 wt cells.


title: clr1D_Rep_3

value: h- clr1D::KanMX4  ade6-210/216?  arg3D  his3D  leu1-32  ura4-D18


title: clr1D_Rep_2


title: Suz12_minusTmx

description: DNA attached to Suz12 in Ezh2 wt cells.

value: m ESC Ezh2 fl/fl


title: clr2D_Rep_1


title: WT_Rep_2

value: PY 42


title: WT_Rep_1


title: hnRNP C

description:
RNA attached to hnRNP C in Ezh2 wt cells
Protein crosslinked RNA
Processed data file: hnRNPC.ensembl59.bed.gz

data_processing:
Fastq files were demultiplexed and aligned using the iCount server
High-confidence peaks were identified using the 'peaks' option on the iCount server, with the parameters regionAsOne, flank 50, random perms 100 and fdr 0.05
Reads were filtered to remove any peaks within 50 nt of a repeatMasker-annotated repeat or a short non-coding RNA
Genome_build: mm9
Supplementary_files_format_and_content: BED files containing number of reads in each Ensembl59 gene for that iCLIP in the score column (column 5)

extract_protocol:
Individual-nucleotide resolution UV crosslinking and immunoprecipitation protocols were based in protocols available at Huppertz et al. Cells were irradiated with 0.15 J/cm2 of UV light in a Stratalinker 2400 (Stratagene) with bulbs emitting at 254nm, and 25 x10^6 cells per IP were lysed in 1ml of Lysis buffer (50 mM Tris–HCl, pH 7.4; 100 mM NaCl; 1% Igepal CA-630; 0.1% SDS; 0.5% sodium deoxycholate; plus protease inhibitors). Lysates were syringed with a 27G needle and incubated in a Thermomixer at 37°C and 1100 rpm for three minutes after addition of 200U/ml of DNase Turbo and of 20 U/ml of RNase I. IP was set up using 10 μl of α-hnRNP C (Santa Cruz #32308)  as described in iCLIP protocol. Beads were washed 3x with 900 μl high-salt buffer (50 mM Tris-HCl, pH 7.4; 1 M NaCl; 1 mM EDTA; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate and Urea 1M), 2x with 900 μl wash buffer (20 mM Tris-HCl, pH 7.4; 10 mM MgCl2; 0.2% Tween-20) resuspended in 20 μl PNK mix (15 μl water; 4 μl 5x PNK pH 6.5 buffer [350mM Tris-HCl, pH 6.5; 50m MgCl2 25mM dithiothreitol]; 0.5 μl PNK enzyme; 0.5 μl RNasin [Promega]) and incubated 30 min at 37°C 1100 rpm. Beads were washed 3x with 900 μl high-salt buffer, 2x with 900 μl wash buffer, resuspended in beads in 20 μl ligation mix (9 μl water; 4 μl 4x ligation buffer [200 mM Tris-HCl; 40m MM gCl2; 40 mM dithiothreitol]; 1 μl RNA ligase [NEB]; 0.5 μl RNasin [Promega]; 1.5 μl pre-adenylated linker L3 [20 μM]; 4 μl PEG400 [81170, Sigma]), ligation was performed over night at 16°C and 1100 rpm. Beads were washed 3x with high-salt buffer, 2x wash buffer and 20% of the beads were incubated with 8 μl of hot PNK mix (0.4 μl PNK [NEB]; 0.8 μl 32P-γ-ATP; 0.8 μl 10x PNK buffer [NEB]; 6 μl water), radiolabelled and non-radiolabelled beads were pooled again and resuspended in 20 μl in Nupage loading buffer with reducing agent and run on a 4-12% NuPAGE Bis-Tris gel (Invitrogen) in MOPS buffer according to the manufacturer's instructions. Protein-RNA complexes were transferred to a nitrocellulose membrane (Hybond, GE Healthcare) washed 2x with PBS1x and exposed to a Fuji film. Using the auto-radiograph as a mask RNA-protein complexes attached to the membrane were isolated and treated with 200 μl PK buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA) and 10 μl proteinase K (Roche, 03115828001) for 20 minutes at 1,100 rpm and 37°C. 200 μl of PK-urea buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA; 7 M urea) was added and a second incubation was performed for 20 minutes at 37°C and 1100 rpm. Solution was collected and RNA was isolated by phenol: chloroform extraction and ethanol precipitation over night at -20°C.
RNA pellet was resuspended in 7.25 μl RNA/primer mix (6.25 μl water; 0.5 μl Rclip primer [0.5 uM]; 0.5 μl dNTP mix [10mM]) and transferred in a 0.2 ml tube and incubated at 70°C. Retrotranscription was performed by addition of 2 μl 5x RT buffer; 0.5 μl 0.1M DTT; 0.25 μl Superscript III reverse transcriptase [Invitrogen] and incubation 20 min at 42°C, 40 min at 50°C and 5 min at 80°C. cDNA was fractionated running samples on a precast 6% TBE-urea gel 180V 40 min and isolating bands at 250-180 nt (top), 120-180 nt (high), 85-120 nt (medium) and 70-85 nt (low). Gel slices were submerged and crushed in TE buffer, incubated 1h at 37°C 1100 rpm, flash frozen, and a second incubation was performed 1h at 37°C 1100 rpm. cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C. cDNA was resuspended in 8 μl ligation mix (6.5 μl water; 0.8 μl 10x CircLigase Buffer II; 0.4 μl 50 mM MnCl2; 0.3 μl Circligase II [Epicentre]) transferred into a 0.2 tube and incubated for 1 h at 60°C for circularization. Samples were incubated 30 μl oligo annealing mix (26 μl water; 3 μl FastDigest Buffer [Fermentas]; 1 μl cut_oligo [10 μM]), oligo was annealed by temperature gradient and circular cDNA was re-linearized incubating samples with 2 μl BamHI (Fast Fermentas).
cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C and pellet were resuspended in 20 μl of H20. Amplification was performed using 10 μl cDNA; 1 μl primer mix P5/P3 solexa, 10 μM each; 20 μl Accuprime Supermix 1 enzyme [Invitrogen], 9 μl H20 and the following PCR programme: 94°C for 2 min, [94°C for 15 sec, 65°C for 30 sec, 68°C for 30 sec]n cycles, 68°C for 3 min, 4°C, with a number of cycles optimized previously. Library integrity was analysed using 8 μl PCR product with 8 μl of 5x TBE loading buffer and loading on a precast 6% TBE gel (Invitrogen). Gel was stained with Sybrgreen I (Invitrogen) and analyse with a gel imager. Library concentration was determined using the KAPA Universal Library Quantification kit, according to the manufacturer’s instructions and library concentration was corrected by multiplication by 0.38 to improve cluster density. Libraries were sequenced using Illumina HiSeq 2500 sequencers (50 bp single-end reads).

value: No Tamoxifen

value: hnRNP C Santa Cruz #32308


title: Suz12 Ezh2 d/d 2

description:
RNA attached to Suz12 in Ezh2 KO cells. Replicate2
Protein crosslinked RNA
Processed data file: Suz12_Ezh2dd.ensembl59.bed.gz


title: IgG

description:
RNA attached to IgG in Ezh2 wt cells
Protein crosslinked RNA
Processed data file: IgG.ensembl59.bed.gz

extract_protocol:
Individual-nucleotide resolution UV crosslinking and immunoprecipitation protocols were based in protocols available at Huppertz et al. Cells were irradiated with 0.15 J/cm2 of UV light in a Stratalinker 2400 (Stratagene) with bulbs emitting at 254nm, and 200 x10^6 cells per IP were lysed in 1ml of Lysis buffer (50 mM Tris–HCl, pH 7.4; 100 mM NaCl; 1% Igepal CA-630; 0.1% SDS; 0.5% sodium deoxycholate; plus protease inhibitors). Lysates were syringed with a 27G needle and incubated in a Thermomixer at 37°C and 1100 rpm for three minutes after addition of 200U/ml of DNase Turbo and of 20 U/ml of RNase I. IP was set up using 10 μg of IgG (Santa Cruz #2027)  as described in iCLIP protocol. Beads were washed 3x with 900 μl high-salt buffer (50 mM Tris-HCl, pH 7.4; 1 M NaCl; 1 mM EDTA; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate and Urea 1M), 2x with 900 μl wash buffer (20 mM Tris-HCl, pH 7.4; 10 mM MgCl2; 0.2% Tween-20) resuspended in 20 μl PNK mix (15 μl water; 4 μl 5x PNK pH 6.5 buffer [350mM Tris-HCl, pH 6.5; 50m MgCl2 25mM dithiothreitol]; 0.5 μl PNK enzyme; 0.5 μl RNasin [Promega]) and incubated 30 min at 37°C 1100 rpm. Beads were washed 3x with 900 μl high-salt buffer, 2x with 900 μl wash buffer, resuspended in beads in 20 μl ligation mix (9 μl water; 4 μl 4x ligation buffer [200 mM Tris-HCl; 40m MM gCl2; 40 mM dithiothreitol]; 1 μl RNA ligase [NEB]; 0.5 μl RNasin [Promega]; 1.5 μl pre-adenylated linker L3 [20 μM]; 4 μl PEG400 [81170, Sigma]), ligation was performed over night at 16°C and 1100 rpm. Beads were washed 3x with high-salt buffer, 2x wash buffer and 20% of the beads were incubated with 8 μl of hot PNK mix (0.4 μl PNK [NEB]; 0.8 μl 32P-γ-ATP; 0.8 μl 10x PNK buffer [NEB]; 6 μl water), radiolabelled and non-radiolabelled beads were pooled again and resuspended in 20 μl in Nupage loading buffer with reducing agent and run on a 4-12% NuPAGE Bis-Tris gel (Invitrogen) in MOPS buffer according to the manufacturer's instructions. Protein-RNA complexes were transferred to a nitrocellulose membrane (Hybond, GE Healthcare) washed 2x with PBS1x and exposed to a Fuji film. Using the auto-radiograph as a mask RNA-protein complexes attached to the membrane were isolated and treated with 200 μl PK buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA) and 10 μl proteinase K (Roche, 03115828001) for 20 minutes at 1,100 rpm and 37°C. 200 μl of PK-urea buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA; 7 M urea) was added and a second incubation was performed for 20 minutes at 37°C and 1100 rpm. Solution was collected and RNA was isolated by phenol: chloroform extraction and ethanol precipitation over night at -20°C.
RNA pellet was resuspended in 7.25 μl RNA/primer mix (6.25 μl water; 0.5 μl Rclip primer [0.5 uM]; 0.5 μl dNTP mix [10mM]) and transferred in a 0.2 ml tube and incubated at 70°C. Retrotranscription was performed by addition of 2 μl 5x RT buffer; 0.5 μl 0.1M DTT; 0.25 μl Superscript III reverse transcriptase [Invitrogen] and incubation 20 min at 42°C, 40 min at 50°C and 5 min at 80°C. cDNA was fractionated running samples on a precast 6% TBE-urea gel 180V 40 min and isolating bands at 250-180 nt (top), 120-180 nt (high), 85-120 nt (medium) and 70-85 nt (low). Gel slices were submerged and crushed in TE buffer, incubated 1h at 37°C 1100 rpm, flash frozen, and a second incubation was performed 1h at 37°C 1100 rpm. cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C. cDNA was resuspended in 8 μl ligation mix (6.5 μl water; 0.8 μl 10x CircLigase Buffer II; 0.4 μl 50 mM MnCl2; 0.3 μl Circligase II [Epicentre]) transferred into a 0.2 tube and incubated for 1 h at 60°C for circularization. Samples were incubated 30 μl oligo annealing mix (26 μl water; 3 μl FastDigest Buffer [Fermentas]; 1 μl cut_oligo [10 μM]), oligo was annealed by temperature gradient and circular cDNA was re-linearized incubating samples with 2 μl BamHI (Fast Fermentas).
cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C and pellet were resuspended in 20 μl of H20. Amplification was performed using 10 μl cDNA; 1 μl primer mix P5/P3 solexa, 10 μM each; 20 μl Accuprime Supermix 1 enzyme [Invitrogen], 9 μl H20 and the following PCR programme: 94°C for 2 min, [94°C for 15 sec, 65°C for 30 sec, 68°C for 30 sec]n cycles, 68°C for 3 min, 4°C, with a number of cycles optimized previously. Library integrity was analysed using 8 μl PCR product with 8 μl of 5x TBE loading buffer and loading on a precast 6% TBE gel (Invitrogen). Gel was stained with Sybrgreen I (Invitrogen) and analyse with a gel imager. Library concentration was determined using the KAPA Universal Library Quantification kit, according to the manufacturer’s instructions and library concentration was corrected by multiplication by 0.38 to improve cluster density. Libraries were sequenced using Illumina HiSeq 2500 sequencers (50 bp single-end reads).

value: Ezh2 wild type

value: IgG Santa Cruz #2027


title: Suz12 Ezh2 fl/fl 3

description:
RNA attached to Suz12 in Ezh2 wt cells. Replicate3
Protein crosslinked RNA
Processed data file: Suz12_Ezh2flfl.ensembl59.bed.gz