title: E03_plate2_121


title: D11_plate2_184

status: Public on Nov 26 2017

type: SRA

city: Boston

first: Ruben

last: Dries

address: 360 Longwood Ave

department: Computational Biology

country: USA

institute: DFCI

description: single cell RNAseq T cells after G1T28 treatment

library_source: transcriptomic

data_processing:
Sequenced reads were mapped to the human gemome (hg19) by STAR for mapping information.
Estimated transcript counts were retrieved via the pseudo-aligner Kallisto using the mouse Gencode vM1 annotation.
Only single cell samples with at least 100.000 paired-end reads and a minimum of 20% aligning to the transcriptome were retained for further analysis.
PCA combined with density based clustering was performed to remove outlier cells based on a combination of quality metrics.
Genes were considered not expressed if TPM < 1 and were removed if not detected in at least 10% of remaining cells.
The R package SCONE was used to define the optimal normalization approach for the data in a 2 step approach. First we used the plates that contain ERCC spike -ins (plate 3 and plate4) and next we used the most stable genes identified in the previous step.
Density based clustering was used to identify different clusters after initial feature selection by selecting most variable and informative genes.
Pairwise comparisons to identify differentially distributed genes were performed with the R package scDD.
Genome_build: mm9
Supplementary_files_format_and_content: log_normalized_expr_plus1.txt: txt file with log normalized expression counts + 1
Supplementary_files_format_and_content: information_raw_data.xlsx
Supplementary_files_format_and_content: phenodata_samples.txt

molecule: total RNA

organism: Mus musculus

source_name: T cell

growth_protocol: Genetically engineered mouse model (GEMM) of conditional activating mutation that harboring endogenous Kras (KrasLSL-G12D/+) were crossed with p53 conditional knockout Trp53fl/fl. CRE recombinase was induced through intranasal inhalation of 5x106 p.f.u. adeno-Cre (University of Iowa adenoviral core).

extract_protocol:
Live CD45+ and CD3+ cells were sorted in 2ul of Qiagen TCL buffer in 96-well plates
SmartSeq2 protocol with modifications (Trombetta et al., 2014)

treatment_protocol: G1T28, 100 mg/kg, PO, QD

tag: tissue

value: Lung tumor

tag: strain

value: Mixed background

tag: gemm model

tag: cell type

value: tumor infiltrating T cells


title: E02_plate2_113

description: single cell RNAseq control T cells from tumor


title: E01_plate2_105


title: D10_plate2_176


title: D09_plate2_168


title: D08_plate2_160


title: D07_plate2_152


title: D05_plate2_136


title: D06_plate2_144


title: D01_plate2_104


title: C12_plate2_191


title: D04_plate2_128


title: D02_plate2_112


title: C11_plate2_183


title: C10_plate2_175


title: D03_plate2_120


title: C09_plate2_167


title: C08_plate2_159


title: C07_plate2_151


title: C05_plate2_135


title: C04_plate2_127


title: C01_plate2_103


title: C06_plate2_143


title: D12_plate2_192


title: C03_plate2_119


title: B11_plate2_182


title: B12_plate2_190


title: B09_plate2_166


title: B08_plate2_158


title: B10_plate2_174


title: B07_plate2_150


title: B06_plate2_142


title: B04_plate2_126


title: B05_plate2_134


title: B02_plate2_110


title: B01_plate2_102


title: A12_plate2_189


title: B03_plate2_118


title: A09_plate2_165


title: A08_plate2_157


title: A11_plate2_181


title: A05_plate2_133


title: A06_plate2_141


title: A07_plate2_149


title: C02_plate2_111


title: A02_plate2_109


title: H12_plate1_96


title: A03_plate2_117


title: A01_plate2_101


title: H09_plate1_72


title: H08_plate1_64


title: H11_plate1_88


title: H05_plate1_40


title: H06_plate1_48


title: H04_plate1_32


title: H03_plate1_24


title: A10_plate2_173


title: H02_plate1_16


title: G12_plate1_95


title: G11_plate1_87


title: H01_plate1_8


title: G09_plate1_71


title: G08_plate1_63


title: G07_plate1_55


title: G10_plate1_79


title: A04_plate2_125


title: G06_plate1_47


title: G04_plate1_31


title: G05_plate1_39


title: F12_plate1_94


title: G02_plate1_15


title: F11_plate1_86


title: G01_plate1_7


title: H10_plate1_80


title: F09_plate1_70


title: F08_plate1_62


title: H07_plate1_56


title: F07_plate1_54


title: F06_plate1_46


title: F05_plate1_38


title: F02_plate1_14


title: E09_plate1_69


title: F01_plate1_6


title: E08_plate1_61


title: E07_plate1_53


title: E11_plate1_85


title: E06_plate1_45


title: E05_plate1_37


title: E02_plate1_13


title: E04_plate1_29


title: E03_plate1_21


title: E01_plate1_5


title: D12_plate1_92


title: D10_plate1_76


title: D09_plate1_68


title: F10_plate1_78


title: D05_plate1_36


title: D08_plate1_60


title: D06_plate1_44


title: D07_plate1_52


title: F03_plate1_22


title: F04_plate1_30


title: E12_plate1_93


title: D02_plate1_12


title: C12_plate1_91


title: C07_plate1_51


title: E10_plate1_77


title: C10_plate1_75


title: C05_plate1_35


title: C02_plate1_11


title: C03_plate1_19


title: C06_plate1_43


title: B12_plate1_90


title: C09_plate1_67


title: C04_plate1_27


title: C08_plate1_59


title: D11_plate1_84


title: D04_plate1_28


title: B11_plate1_82


title: D03_plate1_20


title: B07_plate1_50


title: B06_plate1_42


title: B10_plate1_74


title: B08_plate1_58


title: B05_plate1_34


title: B03_plate1_18


title: D01_plate1_4


title: B04_plate1_26


title: C11_plate1_83


title: B02_plate1_10


title: A12_plate1_89


title: B01_plate1_2


title: C01_plate1_3


title: A11_plate1_81


title: B09_plate1_66


title: A10_plate1_73


title: A09_plate1_65


title: A05_plate1_33


title: A04_plate1_25


title: A08_plate1_57


title: HT1080_6 mg/mL_1

city: La Jolla

first: Daniel

last: Ortiz

state: California

address: 9500 Gilman Dr

department: Bioengineering

institute: University of California San Diego

data_processing:
Alignment was performed using Bowtie2 and quantification using eXpress pipe line. Reads aligned to the reference human genome Hg19
Genome_build: Hg19
Supplementary_files_format_and_content: eXpress pipeline outputs (fpkm)

organism: Homo sapiens

source_name: 3D collagen cultured human fibrosarcoma cells

growth_protocol: Cells embedded in 3D collagen matrices were prepared by mixing cells suspended in culture medium and 10X reconstitution buffer, 1:1 (v/v), with soluble rat tail type I collagen in acetic acid (Corning, Corning, NY) to achieve the desired final concentration10,22,23. 1 M NaOH was used to normalize pH in a volume proportional to collagen required at each tested concentration (pH 7.0, 10-20 μl 1 M NaOH), and the mixture was placed in 48 well culture plates and let polymerize at 37C. Final gel volumes were 200 uL.

extract_protocol:
3D collagen I gels were seeded in three independent experiments and harvested after 24 hours of culture for RNA extraction and directly homogenized in Trizol reagent (Thermofisher, Waltham, MA). Total RNA was isolated following manufacturer’s instructions. Isolated RNA was further purified using High Pure RNA Isolation Kit (ROCHE, Branford, CT). RNA integrity was verified using RNA Analysis ScreenTape (Agilent Technologies, La Jolla , CA) before sequencing.
TruSeq Stranded mRNA Sample Prep Kit (Illumina, San Diego, CA)

treatment_protocol: No treatment

tag: cell line

tag: culture condition

value: 6 mg/mL collagen matrix


title: MDA-MB-231_2.5 mg/mL_3

source_name: 3D collagen cultured human Breast cancer cells

value: 2.5 mg/mL collagen matrix


title: MDA-MB-231_6 mg/mL_1


title: HT1080_6 mg/mL_3


title: MDA-MB-231_6 mg/mL_3


title: A06_plate1_41


title: A07_plate1_49


title: MDA-MB-231_6 mg/mL_2


title: MDA-MB-231_2.5 mg/mL_1


title: A03_plate1_17


title: HT1080_2.5 mg/mL_3


title: A01_plate1_1


title: HT1080_6 mg/mL_2


title: HT1080_2.5 mg/mL_2


title: A02_plate1_9


title: HT1080_2.5 mg/mL_1


title: HFF_6 mg/mL_3

source_name: 3D collagen cultured human foreskin fibroblast

value: HFF


title: HFF_6 mg/mL_2


title: HFF_6 mg/mL_1


title: MDA-MB-231_2.5 mg/mL_2


title: HFF_2.5mg/mL_1


title: HFF_2.5mg/mL_2


title: LTP3_rna-seq

status: Public on Nov 25 2017

city: Chengdu

first: jin

last: long

address: Hui ming

country: China

institute: Sichuan Agricultural University

data_processing:
Illumina Casava1.82 software was used for basecalling.
High-quality reads were obtained by discarding reads containing over 10% Ns, or overlapping at least 10 bp with adaptor sequences at mismatch rate equal to or less than 10%, or including over 50% bases with base quality score less than five, and then mapped to goat genome using Tophat 2.0.11.
Cufflinks version 2.2.1 were applied to quantify gene expression and obtain FPKM expression values (denoted as the fragments per Kb of transcript per Mb mapped reads).
Genome_build: Sus scrofa 10.2
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample

organism: Sus scrofa

source_name: LDM: longissimus dorsi muscle

growth_protocol: three free-ranging individuals in Tibetan highland with the average altitude of 4,000 m (high-altitude); three counterparts under experimental conditions with normal daily feeding in geographically neighboring low-altitude regions (Chengdu plain) with average altitude of 500 m (low-altitude)

extract_protocol:
The total RNA was extracted using the TrIzol reagent (Life Technologies, Beijing, China) according to the manufacturer’s protocols. Illumina TruSeq RNA Sample Prep Kit was used with 1 ug of total RNA for the construction of sequencing libraries.
The dUTP based strand-specific RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: The longissimus dorsi muscle were sampled rapidly, and immediately frozen in liquid nitrogen

tag: pig breed

value: Tibetan pigs

tag: average altitude

value: longissimus dorsi muscle

tag: Sex

value: female


title: TP2_rna-seq


title: LTP2_rna-seq


title: TP3_rna-seq


title: LTP2-BS-seq

library_source: genomic

data_processing:
Read sequences produced by the Illumina pipeline in FastQ format were first pre-processed through in-house perl scripts.
Firstly, as a subset of reads contained all of part of the 3’ adapter oligonucleotide sequence, every read was scanned for the adapter sequence, and if detected the read was filtered out. Secondly, since some reads had N (unknown base) in their sequences, the percentage of Ns in each read was calculated, and if the percentage of Ns was larger than 10% the read was removed. Thirdly, reads with low quality (PHRED score <= 5, and percentage of the low quality bases >= 50%) were trimmed.
Bismark software (version 0.12.5) was used to perform alignments of bisulfite-treated reads to a reference genome with the default parameters.
Genome_build: Sus scrofa 10.2
Supplementary_files_format_and_content: files format:txt files; file content: Methylation level (methylated cytosines sequenced/total cytosines sequenced) for each cytosine site. The column headers for the Bisulfite-seq processed data are: Chromosome, Position, Strand, mC count, umC count, Methylation Level, Context, Pvalue, Corrected Pvalue.

molecule: genomic DNA

growth_protocol: three free-ranging individuals in Tibetan highland with the average altitude of 4,000 m (high-altitude); three counterparts under experimental conditions with normal daily feeding in geographically neighboring low-altitude regions (Chengdu plain) with average altitude of 500 m (low-altitude). For each group, only one individual was chosen for BS-seq.

extract_protocol:
DNA of each collected tissue was extracted using DNeasy Blood & Tissue Kit (Qiagen).
A total amount of 5.2 microgram genomic DNA spiked with 26 ng lambda DNA were fragmented by sonication to 200-300bp with Covaris S220, followed by end repair and adenylation. Cytosine-methylated barcodes were ligated to sonicated DNA as per manufacturer’s instructions. Then these DNA fragments were treated twice with bisulfite using EZ DNA Methylation-GoldTM Kit (Zymo Research). And the resulting single-strand DNA fragments were PCR amplificated using KAPA HiFi HotStart Uracil + ReadyMix (2X).The library preparations were sequenced on an Illumina Hiseq 2000 platform


title: LTP1_rna-seq


title: ARE-del HET anti-PD1/CD40_4

city: Frederick

first: Jack

last: Chen

state: MD

address: SAIC-Frederick Miller Dr

institute: NIH

data_processing:
All the reads were mapped to mm10 by star aligner.
Subreads was used for the gene count.
EdgeR was for the differetially expressed genes.
Genome_build: mm10
Supplementary_files_format_and_content: txt format, data were normalized by EdgeR.

source_name: Subcutaneous tumor

growth_protocol: Low passage B16F10 melanoma cells were grown in DMEM medium 48 h previous to SQ injection. A total of 2.5 X10^5 cells in 200 ul HBSS were injected subcutaneously in th right flank of mice.

extract_protocol:
Tumors were removed, flash frozen on dry ice, and RNA was harvested using Maxwell 16 LEV simplyRNA tissue kit. Illumina TruSeq v4 RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ng of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: After 7 days of subcutaneous growth, mice received two times a week the following antibodies:  control rat IgG2A, 0.25 mg agonist mAb CD40 (Clone FGK115B3; MTMB-M-1083-4) ascites;  0.25 mg mAb anti-mouse PD1 (clone RPMI-14; MTM-M-R-587-1) purified antibody or a combination of anti-PD1 and anti-Cd40 at 0.25 mg.

value: B16F10 melanoma cells

tag: genotype

value: ARE-del +/-


title: TP1-BS-seq


title: ARE-del HET anti-PD1/CD40_2


title: ARE-del HET anti-CD40_3


title: ARE-del HET anti-PD1/CD40_1


title: ARE-del HET anti-CD40_4


title: ARE-del HET anti-PD1_3


title: ARE-del HET anti-PD1_1


title: ARE-del HET anti-PD1_2


title: ARE-del HET anti-PD1/CD40_3


title: ARE-del HET control IgG2A_4


title: ARE-del HET anti-CD40_2


title: ARE-del HET control IgG2A_3


title: HFF_2.5mg/mL_3


title: ARE-del HET control IgG2A_2


title: ARE-del HET anti-CD40_1


title: WT anti-PD1/CD40_3

value: ARE-del +/+


title: ARE-del HET control IgG2A_1


title: WT anti-PD1/CD40_2


title: TP1_rna-seq


title: WT anti-CD40_3


title: WT anti-PD1/CD40_1


title: WT anti-CD40_2


title: WT anti-PD1_3


title: WT anti-PD1_2


title: WT anti-PD1_1


title: WT control IgG2A_1


title: WT control IgG2A_4


title: WT control IgG2A_3


title: WT control IgG2A_5


title: WT control IgG2A_2


title: Control 4

city: Linkoping

first: Charlotta

last: Enerbäck

state: Sweden

address: Medical faculty

country: Sweden

institute: Linkoping University

data_processing:
Sequence reads from bisulfite-treated EpiQuest libraries were identified using standard Illumina base-calling software and then analyzed using a Zymo Research proprietary analysis pipeline, which is written in Python and used Bismark (http://www.bioinformatics.babraham.ac.uk/projects/bismark/) to perform the alignment. Index files were constructed using the bismark_genome_preparation command and the entire reference genome. The --non_directional parameter was applied while running Bismark. All other parameters were set to default. Filled-in nucleotides were trimmed off when doing methylation calling. The methylation level of each sampled cytosine was estimated as the number of reads reporting a C, divided by the total number of reads reporting a C or T. Fisher’s exact test or t-test was performed for each CpG site which has at least five reads coverage, and promoter, gene body and CpG island annotations were added for each CpG included in the comparison.
The methylation ratio in each site was used to calculate the methylation difference, which was determined by subtracting the value for the reference sample from the sample of interest. The methylation status was classified as ‘hypermethylated’ if the site was 0-33% more methylated than the reference and p<0.05, ‘strongly hypermethylated’ if the site was 33-100% more methylated than the reference and p<0.05, ‘hypomethylated’ if the site was 0-33% less methylated than the reference and p<0.05 and ‘strongly hypomethylated’ if the site was 33-100% less methylated than the reference and p<0.05. Sites that obtained a p-value of >0.05 were considered insignificant. All sites with a significant difference in methylation in their comparisons were considered to be differentially methylated regions.
Genome_build: Hg19
Supplementary_files_format_and_content: txt files with the processed data showing methylation ratio for each sample and differential methylation between the samples

source_name: epidermis

growth_protocol: Tissue samples were obtained as 4 mm punch biopsies of the skin. After incubation with 3.8% sterile ammonium thiocyanate in PBS for 30 minutes, the epidermal layer was separated from the dermal part of the skin.DNA was immediately isolated using the Blood and Tissue DNA extraction kit (QIAGEN, Hilden, Germany).

extract_protocol:
Extraction of DNA performed with Blood and Tissue extraction kit (Qiagen) according to the manufacturer's instructions.
Libraries were prepared from 200-500 ng of genomic DNA digested with 60 units of TaqαI and 30 units of MspI (NEB) sequentially and then extracted with Zymo Research (ZR) DNA Clean  & Concentrator™-5 kit. Fragments were ligated to pre-annealed adapters containing 5’-methyl-cytosine instead of cytosine according to Illumina’s specified guidelines (www.illumina.com). Adaptor-ligated fragments of 150–250 bp and 250–350 bp in size were recovered from a 2.5% NuSieve 1:1 agarose gel (Zymoclean™ Gel DNA Recovery Kit, ZR). The fragments were then bisulfite-treated using the EZ DNA Methylation-Lightning™ Kit (ZR). Preparative-scale PCR was performed and the resulting products were purified (DNA Clean & Concentrator™ - ZR) for sequencing on an Illumina HiSeq.

tag: group

value: Control


title: Control 6


title: Control 5


title: Psoriasis uninvolved 2

value: Psoriasis uninvolved


title: Psoriasis uninvolved 3


title: CD4 TSLP 25 min STAT5 ChIP-Seq

city: Cincinnati

first: Yrina

last: Rochman

state: OH

address: 3333 Burnet Avenue

department: Allergy and Immunology

institute: Cincinnati Children's Hospital Medical Center

description:
ChIP-Seq Illumina platform
BioWardrobe IDs: 4508

data_processing:
ChIP-Seq data analysis was performed using the BioWardrobe (PMID: 26248465 )
Briefly, fastq files from Illumina pipeline were aligned by bowtie (version 1.1.1 PMID: 19261174) with maximum one allowed error in a sequence and no more than one  hit was allowed. MACS2 (version 2.1.0.20140616 PMID: 18798982) were used to estimate fragment size and to find islands of enrichment. MACS2 were used with q-value threshold less than 0.2. To visualize the data, it were uploaded to UCSC genome browser and fragment coverage were used with estimated fragment size from MACS2 output.
Genome_build: GRCm38/mm10
Supplementary_files_format_and_content: csv file includes peak information.

source_name: spleen + lymph nodes

growth_protocol: naïve CD4 cells were purified and grown in complete RPMI medium supplemented with 10% FCS

extract_protocol:
For ChIP cells were fixed with 0.8% formaldehyde for 8 min at RT and DNA was processed and sonicated accordingly to the protocol from Covaris using Covaris S220 sonicator for 4 min. STAT5 ChIP was performed by Digenode protocol for transcriptional factors.
ChIP libraries were prepared by using ThruPLEX DNA kit (Rubicon).

treatment_protocol: aCD3 plate bound (5ug/ml) + aCD28 soluble (2ug/ml) + IFNg (10ug/ml) for 3 days, rest in medium 3 days, stimulation with TSLP (40 ng/ml, R&D) for 25 minutes

tag: mouse strain

value: aCD3 activated 3 days and rested 3 days CD4 cells

tag: chip antibodies

value: STAT5 (R&D Systems, AF1584)


title: upf1-del-rpb1-del-plasm-LEU2-rpb1-mut-H1085Y-CKY691-rep2

city: Edinburgh

first: Vahid

last: Aslanzadeh

address: Mayfield Road

department: Wellcome Trust Centre for Cell Biology

country: United Kingdom

institute: University of Edinburgh

description: Total RNA (rRNA depleted)

data_processing:
Splice junctions were generated with STAR
Read counts were generated with DICEseq
Genome_build: SacCer3
Supplementary_files_format_and_content: tab-delimited text files include read counts and FPKM values for each sample

organism: Saccharomyces cerevisiae

extract_protocol:
For total RNA, log phase cells were pelleted and lysed in a beadbeater (Biospec Products) with Zirconia beads (Thistle Scientific) and phenol, then extracted with phenol:chloroform and precipitated with ethanol.
Directional RNA Library Prep

tag: genotype/variation


title: MKN45-4BI

city: Jinan

first: Han

last: Bo

address: #44, Wenhua Xi Rd

department: Department of Pathology

institute: Shandong University Medical School

description: MKN45 transfected with negative control

data_processing:
After sequencing, the Solexa CHASTITY quanlity filtered reads were harvested as Clean Reads.
The adaptor sequences were trimmed and the adaptor-trimmed-reads (>= 15nt) were left.
miRDeep2 software was used to predict the novel miRNAs with these trimmed reads. Then, the trimmed reads were aligned to merged pre-miRNA databases (known pre-miRNA from miRBase v21 plus the newly predicted pre-miRNAs) using Novoalign software (v2.07.11) with at most one mismatch. Reads (counts < 2) were discarded when calculating the miRNA expression.
In order to characterize the isomiR variability, sequences that matched the miRNA precursors in the mature miRNAs region ±4 nt (no more than 1 mismatch) were accepted as mature miRNA isomiRs, which were grouped according to the 5-prime (5p) or 3-prime (3p) arm of the precursor hairpin.
The numbers of mapped tags that were defined as the raw expression levels of that miRNA. To correct for the difference in tag counts between samples, the tag counts were scaled to TPM (the copy number of transcripts per million) based on the total number of tag aligned
Genome_build: miRBase v21 plus the newly predicted pre-miRNAs
Supplementary_files_format_and_content: Text data file with normalized counts.  A FASTA file with the novel miRNA sequences is available on the series record.

source_name: MKN45 transfected with negative control

extract_protocol:
Total RNA were extracted using Trizol. The RNA quantity and quality were measured by NanoDrop ND-1000. RNA integrity was assessed by standard denaturing agarose gel electrophoresis.
Total RNA of each sample was used to prepare the miRNA sequencing library, which included the following steps: 1) 3'-adaptor ligation; 2) 5'-adaptor ligation; 3) cDNA synthesis; 4) PCR amplification; 5) size selection of ~135-155 bp PCR amplified fragments (corresponding to ~15-35 nt small RNAs).

treatment_protocol:
MKN45/NS cells were cultured in RPMI1640 medium and then transfected with shSCR lentivirus. 24hs later, fresh medium containing 2μg/ml puromycin was added to each well. After two weeks, stable-expression cell lines were established.
MKN45/4BI cells were cultured in RPMI1640 medium and then transfected with shCUL4B lentivirus. 24hs later, fresh medium containing 2μg/ml puromycin was added to each well. After two weeks, stable-expression cell lines were established.

tag: developmental stage

value: poorly differentiated adenocarcinoma cells

value: metastatic paragastric lymph nodes


title: Psoriasis involved 3

value: Psoriasis involved


title: Psoriasis involved 2


title: CD4 medium STAT5 ChIP-Seq

description:
ChIP-Seq Illumina platform
BioWardrobe IDs: 4507


title: MKN45-NS

description: MKN45  stable knockdown CUL4B

source_name: MKN45 stable knockdown CUL4B


title: upf1-del-rpb1-del-plasm-LEU2-rpb1-mut-H1085Y-CKY691-rep1

description: Total RNA (rRNA depleted)


title: WT anti-PD1_4


title: Psoriasis uninvolved 1


title: upf1-del-rpb1-del-plasm-LEU2-rpb1-mut-G1097D-CKY772-rep2

description: Total RNA (rRNA depleted)


title: Psoriasis involved 1


title: upf1-del-rpb1-del-plasm-LEU2-rpb1-mut-G1097D-CKY772-rep1

description: Total RNA (rRNA depleted)


title: upf1-del-rpb1-del-plasm-LEU2-RPB1-CKY690-rep2

description: Total RNA (rRNA depleted)


title: Control 3


title: Control 2


title: DamID ERG::Dam

city: Manchester

first: Andrew

middle: Joshua

last: Lilly

address: Wilmslow Road

department: Stem Cell Haematopoiesis

institute: CRUK Manchester Institute

data_processing:
Sequencing reads were aligned to mm9 genome using the aligner SMALT (https://www.sanger.ac.uk/resources/software/smalt/) with default settings. Reads aligning to multiple loci were discarded. The BioConductor package BayesPeak was used to call peaks from the uniquely aligned reads for control and test samples seperrately, using the default settings. Peaks with a posterior probability > 0.9 were kept for further analysis. Isolated peaks (that do not overlap with a peak in the control sample) were retained for subsequent analysis. We then identified all instances where one or more peaks from the test and control samples overlapped and extend the boundaries of these regions to contain all contiguous signal (irrespective of whether that came from control or test samples). We then calculated the RPKM (number of reads per kilo base per million reads) in the region from the test sample and the control sample respectively and ranked regions by the difference in RPKM between the test and control samples (i.e. ∆RPKM = RPKM_test - RPKM_control). We selected the 10% of regions with the greatest difference and filtered further to discard loci where fold change of test to control, i.e. RPKM_test/RPKM_control, < 2. Peaks in the test sample overlapping with these remaining regions were added to the set of isolated peaks for further analysis.
We applied the Poisson model based scan statistic method (Kulldorff, M.Theory and Methods, 1997; 26:1481-1496) to this set of peaks to find peak-enriched regions in the genome. A 2000bp window was scanned along each chromosome at 400bp increments. At each position of the sliding window, we calculated the number of bases within the window corresponding to a peak. This value, together with length of the window, was used to compute the value of the scan statistic. Monte Carlo sampling was used to establish an approximate distribution of the scan statistic under the null hypothesis. We selected those windows where the p-value of the scan statistic < 0.05 as peak-enriched windows (PEW) and merged contiguous PEWs to generate larger, peak-enriched regions (PERs), and trimmed the 5' and 3' boundaries of each region to remove nucleotides that do not overlap with any peak. PERs were mapped to genome annotation using ENSEMBL (v66) via the BioConductor package annmap, and retaining PERs that overlapped, or within 30kb of a gene. Where multiple genes mapped to a single PER, the closest gene was selected in order to generate a PER_to_gene mapping for each test sample
Genome_build: mm9
Supplementary_files_format_and_content: peak

source_name: endothelial precursor cells (EPCs)

growth_protocol: ES cell–derived endothelial precursor cells were routinely generated by isolating FLK1+ cells from d3 embryonic body differentiations. FLK1+ cells where maintained on gelatin coated plates in liquid blast medium (IMDM supplemented with 10% FCS, P/S, 2mM L-Glutamine, 20μg/ml transferrin, 0.45mM MTG, 25μg/ml AA, 15% D4T conditioned medium, 5ng/ml VEGF and 10ng/ml IL6). After 1 day of liquid blast culture endothelial precursor cells where isolated by sorting for a cKIT+ TIE2+  phenotype.

extract_protocol:
Genomic DNA Purification Kit (Promega) according to manufacturers instructions and DamID samples where prepared as described (Vogel et al. Nat Protoc 2, 1467-1478, 2007)
Multiplexed libraries were prepared from 50ng of the sheared DNA , with 10 cycles of amplification using an NEBNext Ultra DNA Library Prep Kit for Illumina ( New England Biolabs E7370S ) Libraries were quantified  and average sizes calculated on the Agilent Bioanalyser using the High Sensitivity DNA Chips (Agilent Technologies). Accurate quantitation for pooling of libraries for sequencing was done by qPCR using the Kapa Library Quantification Kit for Illumina (Kapa Biosystems).
Multiplexed libraries were sequenced on the HiSeq 2500  in Rapid Mode using On Board Cluster Generation and TruSeq Rapid SBS chemistry ( Illumina)  The sequencing was a custom recipe of a  single read 60bp + 4 dark cycles to overcome the poor sequence diversity at the start of the sequence.  The input was 14pM with a spike in of 1% phiX .

value: ESC derived EPCs

tag: derivative


title: DamID Sox7::Dam


title: Dam ID control (SOX7)

description: SOX7_Dam peaks were called relative 1-WT Dam peaks

value: iDam


title: Control 1


title: T-cell_HiC

status: Public on Nov 24 2017

city: HOUSTON

first: Jianzhong

last: Su

state: TEXAS

address: 1 Baylor Plaza

institute: Baylor college of Medicine

data_processing:
All data was processed using Juicer (Durand et al.2016; Rao et al.2014)
Loops were annotated using HiCCUPS (Durand et al.2016; Rao et al.2014)
Files ending in '_inter_30.hic' were further processed to include only reads with a read quality score above 30
Genome_build: hg19
Supplementary_files_format_and_content: hic

extract_protocol:
One to five million cells were crosslinked with 1% formaldehyde for 10 min at room temperature. After nuclei permeabilization, DNA was digested with MboI, and digested fragments were labeled using biotinylated d-ATP and ligated. After reverse crosslinking, ligated DNA was purified and sheared to a length of ∼400 bp and biotin labeled DNA fragments were pulled down with streptavidin beads and prepped for Illumina sequencing.
Sequencing libraries were constructed according to previous publication Science 326, 289-93 (2009)

value: Human T-cells

tag: cell surface marker


title: HSPC_HiC

value: Human Hematopoietic Stem/Progenitor Cells


title: Erythroid Progenitor_HiC

source_name: CD36+ CD71+ CD235ahi

value: Human Erythroid Progenitor cells

value: CD36+ CD71+ CD235ahi


title: Dam ID control (FLI1/ERG)

description: FLI1/ERG_Dam peaks were called relative 5-WT Dam peaks


title: Glioma EC #3

city: DALLAS

first: KANGSAN

last: KIM

state: TX

address: 5323 Harry Hines Blvd

department: Pathology

institute: UT Southwestern Medical Center

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence(using fastx_trimmer), then mapped to mm10 whole genome using Bowtie2
Read count extraction and nomalization were performed using edgeR.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include read count values for each Sample

source_name: Glioma EC

extract_protocol:
RNA was harvested using Trizol reagent (Invitrogen). 500 ng of total RNA was used for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using LEXOGEN Quant-Seq Library Prep Kit (Cat#001.24) standard protocols.

tag: tissue type

value: Glioma (3weeks after orthotopic injection)

value: Sorted Endothelial cell


title: upf1-del-rpb1-del-plasm-LEU2-RPB1-CKY690-rep1

description: Total RNA (rRNA depleted)


title: Normal brain EC #3

source_name: Normal brain EC

tag: strain

value: normal brain


title: WT anti-CD40_1

value: B16F10 melanoma cells

value: ARE-del +/+


title: Normal brain EC #2


title: HSCP_Canyon_del_HiC

value: Human hematopoietic stem/Progenitor Cells


title: Glioma EC #1


title: Human HSPC_Canyon_deletion_rep3

data_processing:
Reads were aligned to the references genome by hisat2 using default parameters
Gene-level expression measurements for RefSeq genes were reported in fragments per kilobase per million reads (FPKM) by Cufflinks 2.2.1 (http://cufflinks.cbcb.umd.edu/) (Trapnell et al., 2010).
Genome_build: hg19
Supplementary_files_format_and_content: Normalized transcripts as FPKM values

molecule: polyA RNA

source_name: Human HSPC_Canyon_deletion

growth_protocol: Cord Blood CD34+ HSPCs are isolated and placed in the CD34+ expansion medium supplemented with 100ng/mL human SCF, 100ng/mL human FLT3 and 100ng/mL human TPO. One day after culture expansion, CD34+ HSPC is electroporated with Cas9 RNP complex and placed into CD34+ expansion medium. 16-24hrs later, Cells are tested for the deletion of desired regions by PCR and RNA is extracted and saved for RNA-seq analysis.

extract_protocol:
RNA extraction by Qiagen Rneasy micro kit
Followed the manufacturer protocol of Illumia TruSeq stranded mRNA Library Preparation kit

tag: tissue

value: Cord Blood

value: CD34+ hematopoietic stem cells (HSPCs)


title: Glioma EC #2

tag: cell type


title: Human HSPC_Canyon_deletion_rep4


title: Normal brain EC #1


title: Human HSPC_Canyon_deletion_rep2


title: DamID Fli1::Dam

tag: cell line


title: Human HSPC_Canyon_deletion_rep1


title: Human HSPC_Control_rep3

source_name: Human HSPC_Control

value: control


title: Human HSPC_Control_rep1


title: Human HSPC_Control_rep2


title: CD34_INPUT

description: Whole cell extract for H3K27ac and H3K27me3 ChIP-Seq

data_processing:
Sequenced reads were aligned to HG19 referenece genome using bowtie2 with default parameters with the exception of '-k 1'
Peaks were called using MACS2 with  p-val =1e-9 and background datasets as described in characteristic: input. For H3K27me3, the broadPeaks parameter was used.
Genome_build: hg19
Supplementary_files_format_and_content: Processed ChIP-Seq data files are in bigWig format

source_name: Human CD34+ HSPCs

growth_protocol: Cord Blood CD34+ HSPCs are isolated using flow cytometry sorting.

extract_protocol:
CD34+ cells were cross linked with 1% formaldehyde at room temperature for 10 min, and the reaction was stopped by 0.125M glycine at RT 5 min. Cross linked cells were  lysed in 50μl lysis buffer (10 mM Tris pH 7.5, 1mM EDTA, 1% SDS), then diluted with 150 μl of PBS/PIC, and sonicated using Bioruptor (Diagenode) 30sec on 30 sec off 7 cycles to 200-500 bp fragments. ChIP-qualified CTCF (Cell signaling #2899), H3K27ac (abcam #ab4729) and H3K27me3 (Millipore #07-449) antibodies were added to the sonicated chromatin and incubated at 4°C overnight. Previously washed 10 μl of protein A magnetic beads (Invitrogen #88846) were added and incubated for additional 2 hours at 4°C. Immunoprecipitated complexes were washed three times with RIPA buffer and twice with TE (10 mM Tris pH 8.0/1 mM EDTA) buffer. Following transfer into new 1.5 ml collection tube, genomic DNA was eluted for 2 hours at 68 °C in 100 µl Complete Elution Buffer (20 mM Tris pH 7.5, 5 mM EDTA, 50 mM NaCl, 1% SDS, 50 µg/ml proteinase K), and combined with a second elution of 100 µl Elution Buffer (20 mM Tris pH 7.5, 5 mM EDTA, 50 mM NaCl) for 10 min at 68 °C. ChIPed DNA was purified by MinElute Purification Kit (Qiagen #28006) and eluted in 12 µl elution buffer.
Chip-Seq libraries was constructed using Rubicon Genomics ThruPLEX® DNA-seq Kit


title: CD34_CTCF_INPUT

description: Whole cell extract for CTCF ChIP-Seq


title: CD34_CTCF

description: CTCF ChIP-Seq in CD34 hematopoietic stem cells

tag: input

tag: chip antibody

value: CTCF

tag: chip antibody vendor

value: Cell signaling

tag: chip antibody cat. #


title: CD34_H3K27ac

description: H3K27ac ChIP-Seq in CD34 hematopoietic stem cells

value: abcam


title: Zfp281KO_E6.5_embryo_rep4

city: New York

first: Jianlong

last: Wang

state: New York

department: Department of Medicine

institute: Columbia University Irving Medical Center

description: Post-implantation embryos at E6.5

data_processing:
Reads were aligned to the genome using TopHat (v2.0.10) and Bowtie2 (v2.1.0) with the default parameter settings.
Transcript assembly and differential expression analysis were performed using Cufflinks (v2.1.1). Assembling of novel transcripts was not allowed (-G), other parameters of Cufflinks followed the default setting.
Genome_build: mm9
Supplementary_files_format_and_content: tsv files - tab-delimited files containing FPKM values for RefSeq genes in each sample generated using Cufflinks.

growth_protocol: Post-implantation embryos were dissected in DMEM/F12 media supplemented with 5% newborn calf serum.

extract_protocol:
Total RNA was isolated using the TRIZOL reagent (Ambion, #15596018) according to manufacturer’s instructions.
10~100 ng total RNA from each embryo was processed for RNA-seq library preparation using the Ovation Mouse RNA-seq kit (NuGEN, #0348-32) followed the manufactory protocol, then massively parallel sequencing was performed with the Illumina HiSeq 4000 Sequencing System. 100-bp single-end reads were obtained per sample.

tag: strain background

value: Whole embryo


title: CD34_H3K27me3

description: H3K27me3 ChIP-Seq in CD34 hematopoietic stem cells

value: Millipore


title: Zfp281KO_E6.5_embryo_rep2

description: Post-implantation embryos at E6.5


title: Zfp281KO_E6.5_embryo_rep3

description: Post-implantation embryos at E6.5


title: Zfp281KO_E6.5_embryo_rep1

description: Post-implantation embryos at E6.5


title: WT_E6.5_embryo_rep4

description: Post-implantation embryos at E6.5

value: wild type


title: WT_E6.5_embryo_rep3

description: Post-implantation embryos at E6.5


title: WT_E6.5_embryo_rep1

description: Post-implantation embryos at E6.5


title: f DOb48A m3

city: Denver

first: Philippa

last: Marrack

state: CO

address: 1400 Jackson St

department: Biomedical Research

institute: National Jewish Health

description: T cell receptor alpha chain mRNA sequences

data_processing:
For 454 sequences, basecalling, trimming, and filtering was performed by the Roche 454 GS Run Processor software.  For Ion Torrent sequences, basecalling, trimming, filtering, and barcode demultiplexing was done using the Torrent Suite software with base-recalibration turned off.
Data were analyzed by in-house created software.  The software identified TRAVs and TRAJs as published for mice by IMGT with modifications made because of our own sequencing of T cell receptor alpha chain cDNAs from C57BL/6 mice.  CDR3 regions were identified from the conserved cysteine near the C terminal of the TRAVs to the conserved phenyl alanine/leucine/tryptophan glycine in the TRAJs.  Sequences in CDR3 regions were corrected using in-house generated software.  The software identified TRAV sequences downstream of the conserved cysteine and corrected bases providing that downstream of the base were a string of at least 3 bases that corresponded to a known TRAV sequence.  Likewise the software identified TRAJ sequences upstream of the conserved phenyl alanine/leucine/tryptophan and corrected base misreads if there were a string of at least 3 bases upstream of the misread that corresponded to a known TRAJ sequence.   The first applied in-house program was written in Pascal, the second applied program was written in Python.  Both programs are freely available on the lab website https://nationaljewish.org/research-science/programs-depts/biomedical-research/labs/kappler-marrack-research-lab/.
Barcodes for the 454 sequences were identified using the in-house program described above.  In analyses in which more than one sample were simultaneously sequenced, up to 2 mismatches in the barcodes were allowed.  Bar codes were sufficiently different to allow this correction without ambiguity and overlap between the samples.
For samples with an intial data file containing more than a million reads, the number of reads analyzed was reduced to 950,000 by taking the first reported 950,000 reads.
Misreads in TRAV and TRAJ sequences were not corrected since the genes for these segments of T cell receptors do not mutate.
Genome_build: NA
Supplementary_files_format_and_content: gzipped tab-separated text; list of all sequences used in the analysis, with assignment of V- and J-families and CDR3 sequences, etc.

source_name: Naïve CD4 T cells from lymph nodes

extract_protocol:
T cells were stained and sorted at the National Jewish Health Flow Cytometry Core Facility and stored at -80°C as pellets. RNA was isolated using the RNeasy Kit (Invitrogen). cDNA was made using the SuperScript VILO Kit (Invitrogen).
1st PCR:  The cDNA is used as a template for a PCR reaction which pools all of the positive strand TCR Valpha oligos.   Each PCR reaction uses a Cα primer, negative strand (see Silberman & al. (2016), PNAS 113(38):E5608-17, doi: 10.1073/pnas.1609717113 for primer sequences).  The positive strand oligo is specific for 20 different Vα’s (some families require more than one oligo) and adds suitable adapters for either 454 or Ion Torrent sequencers. All PCR products are separated by size on a 1.5% TBE agarose gel and excised from the gel in the range of 250 bp- 500 bp. 2nd PCR: A reaction using 2.5 ul of the pooled bands is prepared.   The positive primer binds to the sequencing adapter.  The negative primer is just 5’ of the Cα oligo used in the first PCR and adds a bar code which denotes the cell sample used. Products are run on a 1.5% TBE gel and the wide band (500-700 bp) is excised from the gel and extracted using a Qiaquik kit without heating.

tag: genotype

value: B6, MHCf, TCRb-/-, Tg DOb48A+, TCRA+/-


title: f DObWT m1

value: B6, MHCf, TCRb-/-, Tg DObWT+, TCRA+/-


title: b DOb48A m3

value: B6, MHCb, TCRb-/-, Tg DOb48A+, TCRA+/-


title: s DOb48A m1

value: B6, MHCs, TCRb-/-, Tg DOb48A+, TCRA+/-


title: Human HSPC_Control_rep4


title: s DOb48A m3


title: b DOb48A m1


title: b DOb48A m2


title: L_WT_J12

city: Montpellier

first: Tristan

last: Bouschet

address: 141 rue de la cardonille

department: CNRS 5203, INSERM U661

country: France

institute: Institut de Génomique Fonctionnelle

data_processing:
Image analyses and base calling were performed using the HiSeq Control Software and Real-Time Analysis component.
Demultiplexing was performed using bcl2fastq 2.18 (Illumina).
Alignment was performed with TopHat 2.1.1 (with Bowtie 2.2.9) to the mm9 version of the Mus muculus genome. The transcript annotation was retrieved from UCSC on January 15, 2012.
Counting of reads aligned to a gene was performed with HTSeq-count 0.6.1p1.
Genome_build: mm9
Supplementary_files_format_and_content: Tab-separated text file. First column is the gene identifier (Gene symbol|Entrez gene ID), the following columns are the raw counts (non-normalized) for each sample.

source_name: mouse embryonic fibroblasts from E13.5 embryos

growth_protocol: Mouse embryonic fibroblasts from wildtype and Zac1-mutant E13.5 embryos were grown in DMEM/FBS10% at 37°C in a 5%CO2 humidificated incubator according to standard procedures

extract_protocol:
Total RNAs were prepared with RNAnow reagent, according to Ozyme's instructions, and DNase treated according to Ambion's protocol.
RNA-Seq libraries were prepared with the 'TruSeq Stranded RNA sample preparation Kit' (Illumina) according to the manufacturer's instructions.

treatment_protocol: Proliferative cells were harvested after 2 days in vitro and quiescent cells after 8, 10 and 12 days in vitro.

value: Zac+/+

value: mouse embryonic fibroblasts

tag: day in vitro

tag: biological replicate

value: 2 ('L' wildtype line)


title: WT alpha 3

source_name: Naïve CD4 T cells from spleen


title: elav-GAL4_H1_n4

city: Hobart

first: Owen

last: Marshall

state: Tas

address: 17 Liverpool St

country: Australia

institute: Menzies Institute for Medical Research

description: DamID

data_processing:
FASTQ files were processed with damidseq_pipeline, default options except for H1 datasets, using either version 1.3 (wor-GAL4 datasets) or version 1.2.6 (other datasets).  Histone H1-vs-Dam samples were generated with the  --norm_method=rpm option.  Biological replicates in gatc.bedgraph format were averaged, and the averaged binding tracks used for further analysis.  Datasets were scaled by dividing each dataset by its standard deviation.
Processed data from the damidseq_pipeline software in gatc.gff format for the five chromatin proxy proteins was merged, sorted by chromosome and location, and a gaussian Hidden Markov Model resolved using the RHmm R package with 2000 random starts and 5 to 15 iterations per start. A Viterbi path was then generated from the best fitting model and the data using the RHmm R package. Each cell type was modeled separately to allow for the possibility that not all chromatin states would be shared between cell types.    Chromatin states were then assigned broad chromatin classes with reference to similarities in protein binding, hclust clustering of state means, and HMM state transition probabilities . States with similar mean protein binding and a high shared emission probability were considered to be related. A simplified Viterbi path was generated from these merged states, and used for subsequent analysis.
Genes were assigned chromatin states based on the modal state. To allow for the large number of short gene bodies in the Drosophila genome and the GATC resolution of the data the modal state was assigned as the state occupying the majority of each gene body + 250bp in each direction. R scripts for the complete data analysis pipeline are available upon request.
Genome_build: DMBDGP6
Supplementary_files_format_and_content: gatc.bedgraph files were generated using the damidseq_pipeline software, at GATC fragment resolution.  Score represents the normalised log2(Dam-fusion/Dam) enrichment.  Processed files are provided as both raw data and scaled data.

organism: Drosophila melanogaster

source_name: whole adult heads 2 days post eclosure

extract_protocol:
DNA was extracted from the anterior half of torn larvae via Qiagen Blood and Tissue kits as per manufacturers' instructions, digested with DpnI, DamID adaptors ligated, digested with DpnII and amplified via PCR.
DNA was sonicated to an average size of 300bp using a Diagenode Bioruptor and DamID adaptors removed via either AlwI or Sau3AI digestion.  Sonnicated material was end-repaired, A-tailed, sequencing adaptors ligated and PCR amplified.

value: brain

value: adult


title: L_KOpat_J10

value: Zac+/-

value: 2 ('L' Zac+/- line)


title: L_KOpat_J12


title: WT alpha 2


title: elav-GAL4_PolII_n4

description: DamID


title: elav-GAL4_Dam_n2

description: DamID


title: elav-GAL4_PolII_n1

description: DamID


title: elav-GAL4_PolII_n2

description: DamID


title: elav-GAL4_Dam_n4

description: DamID


title: L_WT_J10


title: EP_WGBS

description: EP Whole Genome  Bisulfite Sequencing

data_processing:
use BSMAP-2.90 to align bisulfite-treated sequencing reads to the human genome hg19 of bisulfite treated data
use the module of Bsratio of BSMAP-2.90 to compute the methylation ratio of single base level
Genome_build: hg19
Supplementary_files_format_and_content: Chromosome, location, strand, context, ratio, eff_CT_count, C_count, CT_count, rev_G_count, rev_GA_count, CI_lower, CI_upper

source_name: Erythroid Progenitor

growth_protocol: CD34+ CD38- progenitors were cultured in 70% IMDM, 15% FBS, 15% human serum supplemented with 50ng/mL human SCF, 2U/mL human EPO and 2ng/mL human IL3. Culture medium was then replaced by the same base medium with 25ng/mL SCF, 2U/mL EPO on day 3 and 10ng/mL SCF, 2U/mL EPO on day 6. CD36+ CD71+ CD235ahi erythroid progenitors were sorted

extract_protocol:
DNA extraction by Purelink extraction kit
bisulfite treated DNA was made into library with Illumina TruSeq DNA sample preparation kit

value: Human Erythroid Progenitor Cells

value: CD36+ CD71+ CD235ahi


title: elav-GAL4_Dam_n1

description: DamID


title: WT alpha 1


title: f DObWT

source_name: CD4+ CD8+ TCR- preselection thymocytes


title: WT_E6.5_embryo_rep2

description: Post-implantation embryos at E6.5

tag: genotype/variation


title: L_KOpat_J8


title: elav-GAL4_H1_n2

description: DamID

tag: developmental stage


title: elav-GAL4_H1_n1

description: DamID


title: elav-GAL4_Dam_n3

description: DamID


title: L_WT_J8


title: elav-GAL4_HP1a_n4

description: DamID


title: B6


title: L_KOpat_J2


title: elav-GAL4_HP1a_n3

description: DamID


title: b DObWT

value: B6, MHCb, TCRb-/-, Tg DObWT+, TCRA+/-


title: elav-GAL4_HP1a_n2

description: DamID


title: I_KOpat_J12

value: 1 ('I' Zac+/- line)


title: b DOb48A


title: elav-GAL4_Pc_n3

description: DamID


title: I_WT_J12

value: 1 ('I' wildtype line)


title: I_WT_J10


title: G03_plate1_23

data_processing:
Sequenced reads were mapped to the human gemome (hg19) by STAR for mapping information.
Estimated transcript counts were retrieved via the pseudo-aligner Kallisto using the mouse Gencode vM1 annotation.
Only single cell samples with at least 100.000 paired-end reads and a minimum of 20% aligning to the transcriptome were retained for further analysis.
PCA combined with density based clustering was performed to remove outlier cells based on a combination of quality metrics.
Genes were considered not expressed if TPM < 1 and were removed if not detected in at least 10% of remaining cells.
The R package SCONE was used to define the optimal normalization approach for the data in a 2 step approach. First we used the plates that contain ERCC spike -ins (plate 3 and plate4) and next we used the most stable genes identified in the previous step.
Density based clustering was used to identify different clusters after initial feature selection by selecting most variable and informative genes.
Pairwise comparisons to identify differentially distributed genes were performed with the R package scDD.
Genome_build: mm9
Supplementary_files_format_and_content: log_normalized_expr_plus1.txt: txt file with log normalized expression counts + 1
Supplementary_files_format_and_content: information_raw_data.xlsx
Supplementary_files_format_and_content: phenodata_samples.txt

source_name: T cell

extract_protocol:
Live CD45+ and CD3+ cells were sorted in 2ul of Qiagen TCL buffer in 96-well plates
SmartSeq2 protocol with modifications (Trombetta et al., 2014)

value: Lung tumor

value: Mixed background

tag: gemm model

value: tumor infiltrating T cells


title: elav-GAL4_HP1a_n1

description: DamID


title: b DObWT m2


title: elav-GAL4_Pc_n2

description: DamID


title: L_WT_J2


title: elav-GAL4_Pc_n4

description: DamID


title: s DObWT m2

value: B6, MHCs, TCRb-/-, Tg DObWT+, TCRA+/-


title: b DObWT m3


title: I_KOpat_J10


title: b DObWT m1


title: I_KOpat_J8


title: elav-GAL4_Pc_n1

description: DamID


title: f DObWT m3

value: B6, MHCf, TCRb-/-, Tg DObWT+, TCRA+/-


title: I_WT_J8

value: Zac+/+


title: elav-GAL4_Brm_n4

description: DamID


title: s DOb48A

value: B6, MHCs, TCRb-/-, Tg DOb48A+, TCRA+/-


title: f DObWT m2


title: I_KOpat_J2

value: mouse embryonic fibroblasts


title: elav-GAL4_Brm_n2

description: DamID

value: brain


title: f DOb48A m1

value: B6, MHCf, TCRb-/-, Tg DOb48A+, TCRA+/-


title: I_WT_J2


title: elav-GAL4_Brm_n1

description: DamID

value: adult


title: mEpiSC_ChIPseq_Input

description: OEC2 mouse epiblast stem cells grown under primed EpiSCs culture conditon (Fgf+Activin)

data_processing:
FastQC (v0.10.1) was used to check the sequencing quality
Reads were aligned to the mouse genome (NCBI build 37, mm9) using the bowtie (v1.0.0) program, with parameters -m 1.
The mapped reads were sorted and converted to a binary bam file using samtools (v0.1.19)
Zfp281 ChIP-seq peaks were determined by MACS program (v.2.0.10), using input ChIP-seq as the control data, and all other parameters followed the default settings.
Genome_build: mm9
Supplementary_files_format_and_content: Bed file of MACS called peaks.

source_name: Epiblast stem cells

growth_protocol: Primed epiblast stem cells (EpiSCs) were culture on fibronectin-coated plates (10 μg/mL/cm2) in N2B27 medium supplemented with Activin A (20 ng/mL) and Fgf2 (12 ng/mL).

extract_protocol:
ChIP was performed as previously described (Lee et al., 2006) by using Zfp281 antibody (Abcam #ab101318).
25 μl of 70 ng/μl input and 5 ng/μl Zfp281-ChIPed DNA to prepare input and Zfp281 ChIP-seq libraries. Massively parallel sequencing was performed with the Illumina HiSeq 2500 Sequencing System according to the manufacturer’s protocol.

value: mEpiSC OEC2

value: Epiblast stem cells


title: GMR71C09-GAL4_Dam_n3

description: DamID

source_name: dissected 3rd instar larval brain

value: 3rd instar larvae


title: Immunoprecipitated chromatin replicate 2

description: Chromatin IP against Zac1

data_processing:
Image analysis, base calling and alignment were performed using the Illumina pipeline version 1.3.
Peaks were detected and annotated using Cisgenome algorithm v.1 (http://www.biostat.jhsph.edu/~hji/cisgenome/)
Genome_build: mm9
Supplementary_files_format_and_content: Cisgenome output in excel format; Column1: peak rank  Col2: chromosome  Col3: peak start  Col4: peak end  Col5: peak strand (currently all +)  Col6: peak length  Col7: minFDR = minimal false discovery rate for all W bp windows within the peak  Col8: minFDR_pos = the center of the W bp window in which minFDR is obtained  Col9: max|FC| = maximal |log2(fold change)| within a W bp window  Col10: max|FC|_pos = the center of the W bp window in which max|FC| is obtained  Col11: pos_read_num = number of ChIP reads within the peak  Col12: neg_read_num = number of control reads within the peak  Col13-22: information for 5' reads. Col13-18 are counterparts to col7-12.   Col19: Fmaxpos_readnum = maximal number of 5' ChIP reads within a W bp window.   Col20: Fmaxpos_readnum_pos = the center of the W bp window in which the Fmaxpos_readnum was obtained.  Col21: Fmaxneg_readnum = maximal number of 5' control reads within a W bp window.   Col22: Fmaxneg_readnum_pos = the center of the W bp window in which the Fmaxneg_readnum was obtained.  Col23-32: information for 3' reads. They are counterparts to col13-22.  Col33: Rmode-Fmode = distance between the 5' and 3' peaks.  Col34: Delta = {2^min(5' max|FC|, 3' max|FC|)}/2^{max(5' max|FC|, 3' max|FC|)}  Col35: maxPosN = maximal number of ChIP reads within a W bp window.  Col36: maxPosN_pos = the center of the W bp window in which maxPosN was obtained.   Col37: maxNegN = maximal number of control reads within a W bp window.  Col38: maxNegN_pos = the center of the W bp window in which maxNegN was obtained.http://www.biostat.jhsph.edu/~hji/cisgenome/index_files/outputformat_twosamplechipseq.txt

source_name: Zac1 transfected Neuro-2A neuroblastoma cell line

growth_protocol: Neuro-2A cells were grown in DMEM/FBS10%, at 37°C in a 5%CO2 humidificated incubator according to standard procedures.

extract_protocol:
16-hours after transfection, cells were fixated using paraformaldehyde, lysated and nuclei were prepared and sonicated. ChIP was performed with an in-house antiZac1 antibody. The protocol described by Kouskouti & Kyrmizi (http://www.epigenome-noe.net/WWW/researchtools/protocol.php?protid=10) was used.
Libraries were prepared according to Illumina's instructions using the ChIPseq DNA sample prep kit (IP-102-1001). For each sample, 10ng of DNA was repaired, resulting in blunt end fragments and adenylated on its 3' ends. Illumina's adapters were ligated on adenylated DNA. A sizing step was performed on an agarose gel (fragment size including adapters: 200bp) followed by a 18 cycles PCR to amplify fragments with both adapters. Libraries were validated on a DNA1000 Chip on a Bioanalyzer (Agilent).

treatment_protocol: Neuro-2A cells were transfected with pRK5-Zac1 using Lipofectamine according to Invitrogen's instructions.

value: Swiss Albino

value: neuroblastoma Neuro-2A

tag: antibody


title: 18 hpi rep4

city: Ann Arbor

first: Alon

last: Kahana

state: MI

address: 1000 Wall St.

department: Ophthalmology and Visual Sciences

institute: University of Michigan

description: ribosomal depleted total RNA

data_processing:
We checked the quality of the raw reads data for each sample using FastQC (version 0.11.3) to identify features of the data that may indicate quality problems (e.g. low quality scores, over-represented sequences, inappropriate GC content, etc.). We used the software package Tuxedo Suite for alignment, differential expression analysis, and post-analysis diagnostics.
We aligned reads to the reference genome followed by the transcriptome (GRCz10) using TopHat (version 2.0.13) and Bowtie (version 2.2.1). We used default parameter settings for alignment, with the exception of: “--b2-very-sensitive” telling the software to spend extra time searching for valid alignments, and “--max-intron-length” of 400 kbs. Novel transcript discovery was not performed, employing “--no-coverage-search” and “--no-novel-juncs” to limit the read mapping to known transcripts.
We used Cufflinks/CuffDiff (version 2.2.1) for expression quantitation and differential expression analysis, using NCBI GRCz10.fa as the reference genome sequence and NCBI GRCz10.gtf as the reference transcriptome annotation. For this analysis, we used parameter settings: “--multi-read-correct” to adjust expression calculations for reads that map in more than one locus, as well as “--compatible-hits-norm” and “--upper-quartile –norm” for normalization of expression values. We generated diagnostic plots using the CummeRbund package.
Genome_build: GRCz10
Supplementary_files_format_and_content: Processed Data Files include annotated fold-change tab delimited files and differential expression tab delimited files

organism: Danio rerio

source_name: lateral rectus EOM

extract_protocol:
Fish heads were were removed, decalcified in ammonium sulfate EDTA mixture, frozen, cryosectioned and lateral rectus isolated by LMD.  RNA was harvested using Trizol reagent followed by Reliaprep tissue Miniprep system. Illumina TruSeq Stranded mRNA Library Prep Kit and 0.1 to 4 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: lateral rectus EOM

tag: hours post injury


title: B6.AKR

value: B6.AK-H2<k>FlaEgJ mouse


title: mEpiSC_ChIPseq_Zfp281


title: Z12 Zac

data_processing:
Library strategy: DGE-seq
Image analysis and base calling were performed using the Illumina pipeline version 1.3.
Extraction of 16bp and 17pb tags (reads were trimmed for adaptor sequence) and tag counting were performed using home-made perl script.
Tag annotation was performed using home-made perl script.
Genome_build: mm9
Supplementary_files_format_and_content: Tab-delimited file with a first column for gene Symbol and a second column for NCBI GeneID. The next columns correspond to the gene normalized counts (TMM – edgeR)  for each sample.

source_name: Zac1-transfected Neuro-2A

growth_protocol: Neuro2A cells were grown in DMEM/FBS10%, at 37°C in a 5%CO2 humidificated incubator according to standard procedures

extract_protocol:
16-hours after transfection, total RNAs were prepared with TRIZOL reagent, according to Invitrogen's instructions, and DNase treated.
Illumina DGE DpnII Sample preparation kit  (ref.FC-102-1007) was used according to the manufacturer's instruction. Briefly, 2µg of total RNA are incubated with magnetic oligo(dT) beads. Non poly-adenylated RNA are removed by several washes. Reverse transcription is performed on captured RNAs followed by the synthesis of the second strand of cDNA. Captured double stranded DNA is digested using DpnII. A ligation is performed with Illumina's GEX DpnII adapter 1 followed by a digestion using MmeI resulting in the release of tags. Those tags are ligated using Illumina's GEX Adapter 2 , amplified by PCR (15 cycles) and purified on acrylamide gel. Libraries are validated using a DNA1000 BioAnalyzer chip, denatured using 0,1N NaOH, diluted to 8pM and sequenced following the manufacturer's instructions.

treatment_protocol: Neuro-2A were transfected with pRK5-Zac1 and pRK5-CAT, as a negative control, using Lipofectamine according to Invitrogen's instructions.

tag: strain

tag: plasmid


title: C12 CAT

source_name: CAT control-transfected Neuro-2A

tag: cell type


title: MinZ5

data_processing:
Image analysis and base calling were performed using the HiSeq Control Software and Real-Time Analysis component.
Demultiplexing was performed using Illumina's Conversion Software (bcl2fastq 2.17).
The quality of the data was assessed using FastQC from the Babraham Institute and the Illumina software SAV (Sequence Analysis Viewer).
Potential contaminants were investigated with the FastQ Screen software from the Babraham Institute.
RNA-seq reads were aligned to the mouse genome (UCSC mm9) with a set of gene model annotations (genes.gtf downloaded from UCSC on january 15 2012; GeneIDs come from the NCBI: gene2refseq.gz downloaded on january 19 2012), using the splice junction mapper TopHat 2.1.1 (with bowtie 2.2.8).
Final read alignments having more than 3 mismatches were discarded. Gene counting was performed using HTSeq-count 0.6.1p1 (union mode). Since data come from a strand-specific assay, the read has to be mapped to the opposite strand of the gene.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text file include a first column for gene description (Symbol|NCBI GeneID) and next columns correspond to raw abundance measurements (number of reads) for each Sample.

source_name: Zac1-transfected Min6

growth_protocol: Min6 cells were grown in DMEM/FBS15% supplemented with 50mM betaMercapto-ethanol, at 37°C in a 5%CO2 humidificated incubator according to standard procedures

extract_protocol:
16-hours after transfection, total RNAs were prepared with RNAnow reagent, according to Ozyme's instructions, and DNase treated according to Ambion's protocol.
Libraries were prepared according to Illumina's instructions accompanying the Illumina TruSeq Stranded mRNA Sample Preparation Kit.

treatment_protocol: Min6 were transfected with pRK5-Zac1 and pRK5-NLSeGFP, as a negative control, using Lipofectamine according to Invitrogen's instructions.

value: mouse insulinoma Min6


title: MinZ2


title: MinZ1


title: 18 hpi rep5

description: ribosomal depleted total RNA

tag: tissue


title: Z9 Zac


title: MinZ3


title: GMR71C09-GAL4_Dam_n1

description: DamID


title: Input chromatin replicate 2

description: input control


title: B6.NOD


title: C9 CAT


title: GMR71C09-GAL4_Dam_n2

description: DamID


title: 18 hpi rep3

description: ribosomal depleted total RNA


title: MinC5

source_name: control (NLS-eGFP )-transfected Min6


title: GMR71C09-GAL4_PolII_n2

description: DamID


title: s DOb48A m2


title: 18 hpi rep2

description: ribosomal depleted total RNA


title: f DOb48A m2


title: MinC3


title: 18 hpi rep1

description: ribosomal depleted total RNA


title: GMR71C09-GAL4_polII_n1

description: DamID


title: MinC2


title: 9 hpi rep5

description: ribosomal depleted total RNA


title: GMR71C09-GAL4_H1_n2

description: DamID


title: GMR71C09-GAL4_H1_n1

description: DamID


title: 9 hpi rep4

description: ribosomal depleted total RNA


title: GMR71C09-GAL4_HP1a_n2

description: DamID


title: 9 hpi rep3

description: ribosomal depleted total RNA


title: MinC1


title: ARD-16_25_S2

city: Rennes

first: Frédéric

last: Chalmel

state: France

address: 9 avenue du Pr. Léon Bernard

department: Physiology and physiopathology of the urogenital tract

institute: Inserm U1085-Irset

description: Total testis

data_processing:
Base calling. Image analysis and base calling were performed using RTA 1.17.20 and CASAVA 1.8.2.
Assembly of a unique set of human reference transcripts. Ensembl (Cunningham, et al., 2015) and RefSeq (Brown, et al., 2015; Pruitt, et al., 2014) transcript annotations of the hg19 release of the human genome were downloaded from the University of California Santa Cruz (UCSC) genome browser website (Rosenbloom, et al., 2015) in November 4th, 2014. Both transcript annotation files (GTF format) were subsequently merged into a combined set of non-redundant human reference transcripts (HRT) using Cuffcompare (Pollier, et al., 2013). We also defined a non-redundant dataset of human splice junctions (HSJ) extracted from alignments of human transcripts and expressed sequence tags (ESTs) provided by UCSC.
Mapping reads. RNA-seq-derived reads from each sample replicate were aligned independently to the the hg19 release of the human genome sequence with TopHat (version 2.0.10) (Trapnell, et al., 2009) using previously published approaches (Chalmel, et al., 2014; Pauli, et al., 2012; Trapnell, et al., 2012; Zimmermann, et al., 2015). Briefly TopHat program was run a first time for each RNA-seq fastq file using the HRT and HSJ datasets to improve read mapping. The resulting junction outputs produced by all TopHat runs were pooled and added to the HSJ dataset. TopHat was rerun a second time for each sample using the new HSJ dataset. The output of this second run comprised the final alignment (BAM format). Finally, BAM files corresponding to the same isolated testicular cell populations were subsequently merged and sorted with the samtools suite (Li, et al., 2009).
Transcriptome assembly and quantification. The transcriptome of each human testicular cell population was subsequently assembled, compared to known transcript annotation and quantified  with the Cufflinks suite (version 2.2.1) using default settings (Pollier, et al., 2013). Briefly, the assembly step that was performed by Cufflinks using the merged alignment files yielded a set of ~51000-380000 transcript fragments (transfrags) for each testicular cell type. The Cuffcompare program was then used: (i) to define a non-redundant set of 778,012 assembled transcripts by tracking Cufflinks transfrags across all experiments; and, (ii) to compare the resulting transcripts to the HRT dataset (i.e. known transcript annotation). Finally, the abundance of each transcript in each experiment was assessed using Cuffdiff in fragments per kilobase of exon model per million reads mapped (FPKM). Abundance values were quantile normalized to reduce systematic effects and to allow direct comparison between the individual samples.
Refinement of assembled transcripts. As suggested by (Chalmel, et al., 2014; Prensner, et al., 2011), we sequentially applied four filtering steps to eliminate poor-quality quantifications and identify the most robust transfrags from background signal. First, we selected 37,310 “detectable” or “expressed” transfrags defined as those for which abundance levels were above 3 FPKM in at least one testicular cell population. We next selected 33,562 transcripts with a cumulative exon length ≥200 nt. Third, all transfrags that were not automatically annotated by Cuffcompare as complete match (Cuffcompare class “=”), potentially novel isoform (“j”), unknown intronic (“i”, i.e. loci falling entirely within a reference intron and without exon-exon overlap with another known locus), intergenic (“u”) or antisense (“x”) isoforms were discarded, yielding 28,253 transfrags that were retained in the analysis. Finally, all transcript fragments that were annotated as either novel isoforms or novel genes (class codes “j”, “i”, “u” or “x”) and that did not harbor at least two exons (multi-exonic) were filtered out. Together, this strategy produced a high-confidence set of 25,161 transcripts fulfilling these refinement conditions and supporting total RNA molecules expressed in human testicular cells during spermatogenesis
Genome_build: hg19
Supplementary_files_format_and_content: .bed files contain genome-wide read coverage statistics, in bedGraph format, obtained with the "genomeCoverageBed" utility in BEDTools (release 2.12.0); .gtf file contains the reconstructed transcripts obtained with cufflinks and subsequently classified with cuffcompare in the cufflinks suite of tools (release 2.2.1); .FPKM file contains the FPKM expression levels quantified with Cufflinks for each transfrag and each sample; the columns are tab-separated; .xlsx file is an Excel sheet containing all of the information associated with the reconstructed transcripts.

source_name: Total testis

growth_protocol: Human Leydig cells were isolated as previously described (PMID: 3114298) with minor modifications (PMID: 12545077). Briefly, interstitial cells were recovered following a collagenase digestion of the testicular parenchyma (0.5mg/mL, 32°C, 45 minutes) and loaded onto a discontinuous density percoll gradient. After centrifugation at 2500 rpm for 30 minutes, the cells were carefully recovered, washed with PBS and allowed to plate for 2 days at 32°C in DMEM-F12 containing 10% fetal calf serum (FCS), fungizone (2.5µg/ml), penicillin (50 UI/ml), streptomycin (50 µg/ml), vitamin C (0.1 mM), vitamin E (10µg/ml), insulin (10 µg/ml), transferrin (10 µg/ml) and human chorionic gonadotropin (100mUI/ml). The cells were then washed with PBS to remove non-adhering germ cells and were cultured for 3 more days in the same medium without FCS. Cells were then harvested with a cell scraper, pelleted down, snap-frozen and stored at -80°C until RNA extraction.  Human peritubular cells were isolated and cultured as described elsewhere (PMID: 16478819) with minor modifications. First, the testicular parenchyma was submitted to a collagenase digestion (0.5mg/mL, 32°C, 45 minutes). Individualized seminiferous tubule fragments (1-2 cm long) were then selected, placed onto the surface of a plastic cell culture dish and covered with 20-30 µl FCS. The FCS drops were allowed to start evaporating for 30 minutes and DMEM-F12 containing 10% FCS, fungizone (2.5µg/ml), penicillin (50 UI/ml), and streptomycin (50 µg/ml) was then slowly added to the culture dish. After 2 to 3 weeks of culture at 32°C, when peritubular cells had started to grow out of the seminiferous tubules, fragments were removed and the cells were allowed to grow for 2 additional weeks. Cells were then harvested with a cell scraper, pelleted down, snap-frozen and stored at -80°C until RNA extraction.  Isolated human primary Sertoli cells (PMID: 21054948) were purchased from Lonza (Walkersville, USA) and cultured for 3 weeks at 32°C in Sertoli Cell Basal Medium (SeBM™, Lonza) containing FBS, fungizone and gentamicin (SeGM™, Lonza). Cells were then harvested with a cell scraper, pelleted down, snap-frozen and stored at -80°C until RNA extraction.  Pachytene spermatocytes and round spermatids were isolated according to previously described procedures (PMID: 8793064). Briefly, following collagenase digestion of testicular parenchyma (0.5mg/mL, 32°C, 45 minutes), the seminiferous tubules were further dilacerated using scalpels and finally digested again using trypsin (0.3mg/mL, 32°C, 30 minutes). Cell suspensions were next washed with PBS, filtered on nylon gauzes (300, 100 and 20 µm pore size) and on nylon wool prior to separation using an elutriation rotor (JE5 Beckman Instruments, Inc., Fullerton, CA). Cells were then pelleted down, snap-frozen and stored at -80°C until RNA extraction.

extract_protocol:
Total RNA were extracted from tissues and cell pellets using the RNeasy mini Kit (Qiagen), quantified using a NanoDrop™ 8000 spectrophotometer (Thermo Scientific) and quality controlled using a 2100 Electrophoresis Bioanalyzer (Agilent).
Libraries of template molecules suitable for strand specific high throughput DNA sequencing were created using “TruSeq Stranded Total RNA with Ribo-Zero Gold Prep Kit” (catalog # RS-122-2301, Illumina Inc.). Briefly, the removal of cytoplasmic and mitochondrial ribosomal RNA (rRNA) was performed from 500 ng of total RNA using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads. Following purification, the RNA was fragmented using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. The double stranded cDNA fragments were blunted using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. A single ‘A’ nucleotide was added to the 3’ ends of the blunt DNA fragments using a Klenow fragment (3' to 5'exo minus) enzyme. The cDNA fragments were ligated to double stranded adapters using T4 DNA Ligase. The ligated products were enriched by PCR amplification (30 sec at 98°C; [10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C] x 12 cycles; 5 min at 72°C). Excess of PCR primers was removed by purification using AMPure XP beads (Agencourt Biosciences Corporation). Final cDNA libraries were quality-checked and quantified using a 2100 Electrophoresis Bioanalyzer (Agilent). The libraries were loaded in the flow cell at 7pM concentration and clusters were generated in the Cbot and sequenced in the Illumina Hiseq 2500 as paired-end 2x50 base reads following Illumina's instructions.

tag: Sex

value: Male

value: Total testis

value: testis

tag: age

value: Adult


title: ShRP_Ser5P_ChIP-seq

city: Madrid

first: Cian

last: Lynch

state: Comunidad de Madrid

address: C/ Melchor Fernández Almagro, 3

department: Molecular Oncology

country: Spain

institute: Spanish National Cancer Research Centre (CNIO)

description:
ChIP-seq primary mouse embryonic fibroblast; 3 lines pooled.  Control SCR + ChIP-Pol II Ser5P
processed data file: DataAlignedByGene.xlsx

data_processing:
Read files were quality-checked with FastQC.
ChIP-seq reads were aligned to the mouse genome (GRCm38/mm10) using Bwa 0.7.5a
SAMtools 0.1.16 was used to convert the output alignment SAM files to the BAM file format, sort the alignments and eliminate duplicated reads.
BEDTools 2.23.0 was used to convert the resulting files to the BED format. All ChIP and input samples were randomly normalized to the same number of reads.
Peaks were called with MACS 2.0.10.20130712 using the input sample as control for each one of the ChIP samples
genome build: GRCm38/mm10
processed data files format and content: Contain aligned reads per gene

source_name: ChIP-seq BL6 primary mouse embryonic fibroblast; 3 lines pooled

growth_protocol: Cells were maintained in DMEM medium supplemented with 10% FCS plus antibiotics (Pen/Strep)

extract_protocol:
For ChIP-seq assays were performed as described (Rahl et al., 2010, Cell). Briefly, cells were fixed using 1% formaldehyde, harvested, resuspended in ChIP lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1) and sonicated using Covaris water bath sonicator to generate fragments of 150 to 500 bp. Soluble chromatin was diluted 10 fold in ChIP Dilution buffer (1% Triton X-100, 2 mM EDTA pH 8.0, 150 mM NaCl) precleared with Agarose Protein A/G beads (Santa Cruz), and then incuated with antibody specific for total RNA Pol II (N-20, sc-899x, Santa Cruz) or specific for the RNA Pol II Ser5P-phoshorylated form (Abcam #ab5131).  After incubation, immunocomplexes were collected with Agarose Protein A/G beads (Santa Cruz). Next, the immunocomplexes were washed sequentially with Low Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 150mM NaCl), High Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl), LiCl Wash Buffer (0.25M LiCl, 1% NP40, 1% deoxycholate-Na, 1mM EDTA, 10mM Tris-HCl, pH 8.1) and washed twice with TE (10 mM Tris-HCl pH7.5, 1mM EDTA). Immunocomplexes were eluted in ChIP elution buffer (1%SDS, 0.1M NaHCO3) and the crosslinking was reverted by incubation at 65 ºC for 8 hrs with 200 mM NaCl. Samples were treated with Proteinase K and RNase A ,and DNA was extracted using Phenol-Chloroform.  DNA precipitation was in 100% ethanol with 0.1 M NaAcetate ph5.2 and 2 uLs glycogen (Roche).  The DNA pellet was washed with 70% ethanol, and resuspended in ddH2O. Purified chromatin was used for library construction.
For ChIP-seq the amount of DNA used was ~5 ng from each sample (as quantitated by fluorometry). Samples were were processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters as in Illumina's "TruSeq DNA Sample Preparation Guide" (part # 15005180 Rev. C). Adapter-ligated libraries were completed by limited-cycle PCR with Q5 High-Fidelity DNA Polymerase (NEB) and Illumina PE primers (15 cycles), and further purified with a double-sided SPRI size selection to obtain a size distribution in the range of 230-500bp. Libraries were applied to an Illumina flow cell for cluster generation (TruSeq cluster generation kit v5) and sequenced on the Genome Analyzer IIx with SBS TruSeq v5 reagents by following manufacturer's protocols.

treatment_protocol: Cells were harvested at day 3 after  control (SCR) or RPAP1 depletion (shRPAP1 #5) by lentiviral infection.  Selection was maintained in puromycin.

tag: background strain

value: embryonic fibroblast

value: Rb anti Pol II Serine5-phospho (Ser5P), #ab5131, Abcam

tag: treatment

value: RPAP1-knockdown + ChIP-Pol II Ser5P

tag: shRNA

value: shRPAP1 #5


title: ShRP_Pol_II_Total_ChIP-seq

description:
ChIP-seq primary mouse embryonic fibroblast; 3 lines pooled.  Control SCR + ChIP-Pol II total
processed data file: DataAlignedByGene.xlsx

value: Rb anti-Total Pol II (RPB1, N-20) sc-899x, Santa Cruz

value: RPAP1-knockdown  + ChIP-Pol II total


title: GMR71C09-GAL4_Pc_n3

description: DamID

value: brain


title: ARD-15_18_S1

description: Total testis


title: GMR71C09-GAL4_HP1a_n1

description: DamID

value: 3rd instar larvae


title: Immunoprecipitated chromatin replicate 1

description: Chromatin IP against Zac1

value: Swiss Albino


title: ARD-07_5_S1

description: Centrigual elutriation of isolated testicular cells

source_name: Spermatids

value: Spermatides


title: ARD-08_19_S2


title: 9 hpi rep2

description: ribosomal depleted total RNA

tag: genotype

value: lateral rectus EOM


title: 9 hpi rep1

description: ribosomal depleted total RNA


title: Input chromatin replicate 1

description: input control

value: neuroblastoma Neuro-2A


title: GMR71C09-GAL4_Pc_n2

description: DamID


title: SCR_Pol_II_Total_ChIP-seq

value: control SCR + ChIP-Pol II total

value: shSCR non-targeting


title: ARD-06_12_S2

source_name: Spermatocytes

value: Male

value: Spermatocytes


title: ARD-05_6_S1

value: testis

value: Adult


title: GMR71C09-GAL4_Pc_n1

description: DamID


title: 0 hpi rep4

description: ribosomal depleted total RNA


title: SCR_Input_10

description:
ChIP-seq primary mouse embryonic fibroblast; 3 lines pooled.  Control ChIP Input pooled chromatin
input sample; no processed data

value: embryonic fibroblast

value: ChIP Input pooled chromatin, no antibody.

value: control ChIP Input pooled chromatin, no antibody

value: control ChIP Input pooled chromatin


title: 0 hpi rep3

description: ribosomal depleted total RNA


title: ARD-10_25_S2

description: Primary cell culture

source_name: Sertoli cells

value: Sertoli cells


title: GMR71C09-GAL4_Brm_n2

description: DamID

value: brain


title: ARD-09_18_S1

description: Primary cell culture


title: 0 hpi rep2

description: ribosomal depleted total RNA


title: SCR_Ser5P_ChIP-seq

value: Rb anti Pol II Serine5-phospho (Ser5P), #ab5131, Abcam

value: control SCR + ChIP-Pol II Ser5P


title: GMR71C09-GAL4_Brm_n1

description: DamID

value: 3rd instar larvae


title: B-ShRP_RNA-seq

description: RNA-seq#2 wt mouse ES cells replicate B 24 hrs differentiation + RPAP1 depletion shRPAP1#5

data_processing:
Read files were quality-checked with FastQC.
Reads were aligned to the mouse genome (GRCm38/mm10) with TopHat-2.0.4 (using Bowtie 0.12.7 and Samtools 0.1.16), allowing 2 mismatches and 5 multihits.
Transcripts quantification and differential expression calculated with Cufflinks 1.3.0
Mus musculus GRCm38/mm10 transcript annotations from the UCSC Genome Browser were used
genome build: GRCm38/mm10
processed data files format and content: Tables were generated with Cufflinks 1.3.0 and contain transcripts abundance measured in FPKM

source_name: RNA-seq#2 wt mouse ES cells replicate B

growth_protocol: Cells were maintained in standard ES cell Serum/LIF stem cell media during lentiviral infection, then switched to differentiation media (without LIF) for 24 hrs as detailed in the Experimetal Procedures.

extract_protocol:
For RNA-seq Total RNA was extracted using the RNeasy kit (Qiagen)
For RNA-seq, samples of 1 μg of total RNA, with RIN numbers in the range 9.8 to 10 (Agilent 2100 Bioanalyzer), was used.  PolyA+ fractions were processed using TruSeq Stranded mRNA Sample Preparation Kit (Agilent).  Adapter-ligated library was completed by PCR with Illumina PE primers (8 cycles). The resulting purified cDNA library was applied to an Illumina flow cell for cluster generation and sequenced on an Illumina instrument following manufacturer's protocols, for 40 bases in a single-read format (Genome Analyzer IIx, Illumina).  Sequencing quality for RNA-seq samples was analyzed with FastQC. Reads were aligned to the mouse genome (GRCm38/mm10) with TopHat-2.0.4 (Trapnell et al., 2012) (using Bowtie 0.12.7 (Langmead et al., 2009) and Samtools 0.1.16 (Li et al., 2009b), allowing two mismatches and five multihits.  Transcripts assembly, estimation of their abundance, and differential expression, were calculated with Cufflinks 1.3.0 (Trapnell et al., 2012), using the mouse genome annotation data set GRCm38/mm10 from the UCSC Genome Browser (Rosenbloom et al., 2015).

treatment_protocol: Cell treatment was control (SCR) or RPAP1 depletion (shRPAP1 #5) by lentiviral infection and selection in puromycin in standard ES media for self-renewal conditions.  For the experiment, the cells were harvested after differentiated by LIF-removal for 24 hrs, then harvested.

value: ES

tag: library type

value: Strand specific (dUTP)

value: 24 hrs differentiation +RPAP1-knockdown

value: shRPAP1 #5


title: 0 hpi rep1

description: ribosomal depleted total RNA

value: lateral rectus EOM


title: wor-GAL4_Dam_n2

description: DamID


title: XES10876

first: Osvaldo

last: Graña

department: Structural Biology

institute: CNIO

description: Strand-specific RNA-seq (dUTP)

data_processing:
RNA-seq processing:
RNA-seq data was analysed with the nextpresso pipeline as follows:
Reads are quality-checked with FastQC, and aligned to the mouse genome (GRCm38/mm10) with TopHat-2.0.10 (using Bowtie 1.0.0 and Samtools 0.1.19), allowing 2 mismatches and 5 multihits
Differential expression is calculated with DESeq2, using the Mus musculus GRCm38/mm10 transcript annotations from the UCSC Genome Browser
Genome_build: GRCm38/mm10
Supplementary_files_format_and_content: Normalized counts are calculated with DESeq2

source_name: p53 KO T-ALL

growth_protocol: Tissue was extracted and snap-frozen in liquid nitrogen

extract_protocol:
RNA was extracted with the RNeasy Mini Kit (Qiagen #74104) and a DNA digestion step with RNase free Dnase Set (Qiagen #79254) was included.
PolyA+ fraction was purified and randomly fragmented, converted to double stranded cDNA and processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters as in Illumina’s “TruSeq Stranded mRNA Sample Preparation Part # 15031047 Rev. D” kit

treatment_protocol: Not applicable

value: Mixed C57BL/6-129

value: No treatment


title: wor-GAL4_Dam_n1

description: DamID


title: XES10869

description: Strand-specific RNA-seq (dUTP)


title: ARD-14_19_S2

description: Primary cell culture

source_name: Leydig cells

value: Male

value: Leydig cells

value: testis

value: Adult


title: wor-GAL4_PolII_n2

description: DamID


title: A-ShRP_RNA-seq

description: RNA-seq#2 wt mouse ES cells replicate A 24 hrs differentiation + RPAP1 depletion shRPAP1#5

source_name: RNA-seq#2 wt mouse ES cells replicate A


title: B-SCR_RNA-seq

description: RNA-seq#2 wt mouse ES cells replicate B  24 hrs differentiation + control SCR

value: 24 hrs differentiation +control

value: shSCR non-targeting


title: ARD-13_5_S1

description: Primary cell culture


title: XES10862

description: Strand-specific RNA-seq (dUTP)


title: wor-GAL4_PolII_n1

description: DamID

value: brain


title: C-ShRP_RNA-seq

description: RNA-seq#2 wt mouse ES cells replicate C 24 hrs differentiation + RPAP1 depletion shRPAP1#5

source_name: RNA-seq#2 wt mouse ES cells replicate C


title: C-SCR_RNA-seq

description: RNA-seq#2 wt mouse ES cells replicate C 24 hrs differentiation + control SCR


title: shRPAP1_5_RNA-seq

description: RNA-seq#1: primary mouse embryonic fibroblast line#5 RPAP1 depletion shRPAP1#5

source_name: RNA-seq#1: BL6 primary mouse embryonic fibroblast line#5

value: embryonic fibroblast

value: Unstranded


title: GII, Sample_32

city: Balice

first: Kacper

last: Zukowski

address: Krakowska 1 Street

country: Poland

institute: National Research Institute of Animal Production

description:
Sample_32
Processed data file: raw.genes.txt.gz
Processed data file: gene_TE_analysis.txt.gz
Processed data file: sigdiff_gene_TE.txt.gz

data_processing:
The raw sequences were quality controlled using FastQC.
Flexbar software was used to remove Illumina adapters and poly-A sequences.
The raw reads were aligned to the Equus caballus genome (EquCab2.0) as a reference, while the whole procedure was followed by alignment parameters from ENCODE3's STAR-RSEM pipeline.
The quality control metrics across aligned reads per sample were obtained using RNA-SeQC software.
The transposable elements in differential expression (DE) analysis were proceed with Tetranscripts package which utilize DeSeq Bioconductor R-project module to DE analysis.
Genome_build: EquCab2.0
Supplementary_files_format_and_content: raw.genes.txt.gz: compressed gzip file containing raw genes counts.
Supplementary_files_format_and_content: gene_TE_analysis.txt.gz: compressed gzip file containing DE analysis.
Supplementary_files_format_and_content: sigdiff_gene_TE.txt.gz: compressed gzip file containing significant DE genes.

organism: Equus caballus

source_name: blood tissue

extract_protocol:
The RNA was isolated using MagMAX™-96 Total RNA Isolation Kit (Ambion, Life Technologies) according to attached protocol. The quality and quantity of obtained RNA were evaluated on TapeStation 2200 (Agilent Technologies) using RNA Screen Tape and RNA Screen Tape Ladder and by NanoDrop 2000 spectrophotometer (Thermo Scientific). Additionally, RNA was visualized by 2% agarose gel electrophoresis for examining its degradation degree.
The cDNA libraries were prepared from 400ng of total RNA by using TruSeq RNA Kit v2 kit (Cat# FC-122-1001) according to protocol.

tag: breed

value: Arabian horses

value: whole blood

value: 3 years old

tag: gender

value: female

value: racing training


title: wor-GAL4_H1_n1

description: DamID

source_name: dissected 3rd instar larval brain

tag: developmental stage

value: 3rd instar larvae


title: ARD-12_12_S2

description: Primary cell culture

source_name: Peritubular cells

value: Peritubular cells


title: MKJ00159

description: Strand-specific RNA-seq (dUTP)

source_name: CIC LOF T-ALL

value: Mixed C57BL/6-129

value: Tamoxifen-diet from weaning until humane end point.


title: shRPAP1_3_RNA-seq

description: RNA-seq#1: primary mouse embryonic fibroblast line#3 RPAP1 depletion shRPAP1#5

source_name: RNA-seq#1: BL6 primary mouse embryonic fibroblast line#3

value: shRPAP1 #5


title: shRPAP1_1_RNA-seq

description: RNA-seq#1: primary mouse embryonic fibroblast line#1 RPAP1 depletion shRPAP1#5

source_name: RNA-seq#1: BL6 primary mouse embryonic fibroblast line#1


title: MKJ00079

description: Strand-specific RNA-seq (dUTP)


title: ARD-11_6_S1

description: Primary cell culture

value: Male

value: testis


title: GII, Sample_21

description:
Sample_21
Processed data file: raw.genes.txt.gz
Processed data file: gene_TE_analysis.txt.gz
Processed data file: sigdiff_gene_TE.txt.gz

value: male


title: Scr_5_RNA-seq

description: RNA-seq#1: primary mouse embryonic fibroblast line#5 control SCR

value: control

value: shSCR non-targeting


title: MHU00460

description: Strand-specific RNA-seq (dUTP)

source_name: Kras (+/HrasG12V) T-ALL

value: Kras(+/LSLHrasG12V) hUBC-CreERT2(+/T)


title: A-SCR_RNA-seq

description: RNA-seq#2 wt mouse ES cells replicate A  24 hrs differentiation + control SCR

value: ES

value: Strand specific (dUTP)

value: 24 hrs differentiation +control


title: GII, Sample_33

description:
Sample_33
Processed data file: raw.genes.txt.gz
Processed data file: gene_TE_analysis.txt.gz
Processed data file: sigdiff_gene_TE.txt.gz

value: Arabian horses

value: whole blood

value: 3 years old


title: Scr_3_RNA-seq

description: RNA-seq#1: primary mouse embryonic fibroblast line#3 control SCR

value: embryonic fibroblast

value: Unstranded


title: MHU00419

description: Strand-specific RNA-seq (dUTP)

value: Mixed C57BL/6-129


title: wor-GAL4_Pc_n2

description: DamID

value: brain

value: 3rd instar larvae


title: wor-GAL4_H1_n2

description: DamID


title: GI, Sample_52

description:
Sample_52
Processed data file: raw.genes.txt.gz
Processed data file: gene_TE_analysis.txt.gz
Processed data file: sigdiff_gene_TE.txt.gz

value: 2.5 years old

value: untrained


title: Scr_1_RNA-seq

description: RNA-seq#1: primary mouse embryonic fibroblast line#1 control SCR


title: MHU00180

description: Strand-specific RNA-seq (dUTP)

value: Tamoxifen-diet from weaning until humane end point.


title: MKJ00226

description: Strand-specific RNA-seq (dUTP)


title: GI, Sample_51

description:
Sample_51
Processed data file: raw.genes.txt.gz
Processed data file: gene_TE_analysis.txt.gz
Processed data file: sigdiff_gene_TE.txt.gz

value: female


title: wor-GAL4_Pc_n1

description: DamID


title: XES10503

description: Strand-specific RNA-seq (dUTP)

value: No treatment


title: wor-GAL4_Brm_n2

description: DamID


title: GII, Sample_31

description:
Sample_31
Processed data file: raw.genes.txt.gz
Processed data file: gene_TE_analysis.txt.gz
Processed data file: sigdiff_gene_TE.txt.gz

value: racing training


title: wor-GAL4_HP1a_n2

description: DamID


title: MGAS2221 C

city: Reno

first: Paul

last: Sumby

state: NV

address: 1554 N. Virginia St.

department: Microbiology & Immunology

institute: University of Nevada

data_processing:
Rockhopper version 2.03 was used to align sequencing reads to the genome, quantify transcript abundance, and test for differential gene expression. Expression levels correspond to RPKM values except that values are normalized using upper quartile normalization.
Genome_build: For the M1 data we aligned it to the MGAS5005 genome (NC_007297), while for the M3 data we aligned it to the MGAS315 genome (NC_004070).
Supplementary_files_format_and_content: Excel file containing columns for the M3 GAS gene name, M3 GAS gene name synonym, M3 gene product description, M3 GAS gene expression level, corresponding M1 GAS gene columns, q-value, and expression ratio (M3/M1).

organism: Streptococcus pyogenes serotype M1

source_name: MGAS2221 C

growth_protocol: GAS strains were grown in THY broth to the mid-exponential phase of growth (O.D.600 = 0.5).

extract_protocol:
Total RNA was isolated and rRNAs were depleted using the Ribo-Zero Gram-Positive rRNA Removal Kit (Epicentre).
The rRNA depleted RNA was used to generate cDNA libraries using the ScriptSeq kit (Epicentre).  Briefly, RNA was fragmented, cDNA was synthesized using random hexamers containing a 5′ tagging sequence, the RNA was hydrolyzed, and the cDNA was tagged at the 3′ end.  A limited number of PCR cycles (n=12) were used to amplify the libraries via the 5′ and 3′ tags (the libraries were barcoded by using different primers), and the libraries size-selected (170 to 300 bp).  The size-selected and barcoded libraries were run on an Illumina HiSeq flowcell.

value: Parental M1 GAS isolate

value: Exponential phase of growth

tag: growth medium

value: THY broth growth


title: MGAS2221 B

source_name: MGAS2221 B


title: GI, Sample_50

description:
Sample_50
Processed data file: raw.genes.txt.gz
Processed data file: gene_TE_analysis.txt.gz
Processed data file: sigdiff_gene_TE.txt.gz

value: Arabian horses

value: male


title: wor-GAL4_HP1a_n1

description: DamID


title: GI, Sample_49

description:
Sample_49
Processed data file: raw.genes.txt.gz
Processed data file: gene_TE_analysis.txt.gz
Processed data file: sigdiff_gene_TE.txt.gz

value: whole blood


title: MVW415

description: Strand-specific RNA-seq (dUTP)

source_name: Wildtype thymus

value: wildtype


title: GI, Sample_53

description:
Sample_53
Processed data file: raw.genes.txt.gz
Processed data file: gene_TE_analysis.txt.gz
Processed data file: sigdiff_gene_TE.txt.gz


title: MGAS2221 A

source_name: MGAS2221 A


title: GI, Sample_17

description:
Sample_17
Processed data file: raw.genes.txt.gz
Processed data file: gene_TE_analysis.txt.gz
Processed data file: sigdiff_gene_TE.txt.gz


title: MHU367

description: Strand-specific RNA-seq (dUTP)


title: RNG105 cKO, SR

city: Okazaki

first: Rie

last: Ohashi

address: Nishigonaka 38, Myodaiji

country: Japan

institute: National Institute for Basic Biology

data_processing:
Illumina CASAVA version 1.8 software used for basecalling.
Sequence reads were mapped to mouse genome (Mus musculus Ensemble NCBIM37) using TopHat (v 2.0.11) with parameters -p 4 -G.
The mapped reads were assembled by Cufflinks (v 2.2.1), and differential gene enrichment analysis was conducted by Cuffdiff.
Genome_build: Mus musculus Ensemble NCBIM37
Supplementary_files_format_and_content: diff files (tab-delimited text files) include test id, gene id, gene name, locus, test status, FPKM value, log2 (fold change), test statistic value, p value, q value and significant for each sample.

source_name: Hippocampus, SR

extract_protocol:
SP and SR in the CA1 region in the hippocampus were microdissected, and RNA was extracted from the tissues using ISOGEN (Nippon Gene).
Total RNA (200 ng per sample) was used as the starting material to prepare cDNA using TruSeq RNA sample Preparation Kit v2 (Illumina) at a half scale compared with the manufacturer's protocols, as follows. Poly-A-containing mRNAs were purified using Oligo dT magnetic beads. After the mRNAs were denatured and fragmented, first-strand cDNA was synthesized using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific), and then second-strand cDNA was synthesized. After the ends of the fragments were blunted, an adenine nucleotide was added to the 3'-end. RNA adapter index was ligated to the 5'- and 3'-ends of the ds cDNA, and the cDNA was amplified with 10 PCR cycles.

value: hippocampal SR

value: postnatal 12 weeks

value: CaMK2a-Cre; Rng105flox/flox


title: MGAS10870 C

organism: Streptococcus pyogenes serotype M3

source_name: MGAS10870 C

value: Parental M3 GAS isolate


title: s DObWT m1

description: T cell receptor alpha chain mRNA sequences

data_processing:
For 454 sequences, basecalling, trimming, and filtering was performed by the Roche 454 GS Run Processor software.  For Ion Torrent sequences, basecalling, trimming, filtering, and barcode demultiplexing was done using the Torrent Suite software with base-recalibration turned off.
Data were analyzed by in-house created software.  The software identified TRAVs and TRAJs as published for mice by IMGT with modifications made because of our own sequencing of T cell receptor alpha chain cDNAs from C57BL/6 mice.  CDR3 regions were identified from the conserved cysteine near the C terminal of the TRAVs to the conserved phenyl alanine/leucine/tryptophan glycine in the TRAJs.  Sequences in CDR3 regions were corrected using in-house generated software.  The software identified TRAV sequences downstream of the conserved cysteine and corrected bases providing that downstream of the base were a string of at least 3 bases that corresponded to a known TRAV sequence.  Likewise the software identified TRAJ sequences upstream of the conserved phenyl alanine/leucine/tryptophan and corrected base misreads if there were a string of at least 3 bases upstream of the misread that corresponded to a known TRAJ sequence.   The first applied in-house program was written in Pascal, the second applied program was written in Python.  Both programs are freely available on the lab website https://nationaljewish.org/research-science/programs-depts/biomedical-research/labs/kappler-marrack-research-lab/.
Barcodes for the 454 sequences were identified using the in-house program described above.  In analyses in which more than one sample were simultaneously sequenced, up to 2 mismatches in the barcodes were allowed.  Bar codes were sufficiently different to allow this correction without ambiguity and overlap between the samples.
For samples with an intial data file containing more than a million reads, the number of reads analyzed was reduced to 950,000 by taking the first reported 950,000 reads.
Misreads in TRAV and TRAJ sequences were not corrected since the genes for these segments of T cell receptors do not mutate.
Genome_build: NA
Supplementary_files_format_and_content: gzipped tab-separated text; list of all sequences used in the analysis, with assignment of V- and J-families and CDR3 sequences, etc.

source_name: Naïve CD4 T cells from lymph nodes

extract_protocol:
T cells were stained and sorted at the National Jewish Health Flow Cytometry Core Facility and stored at -80°C as pellets. RNA was isolated using the RNeasy Kit (Invitrogen). cDNA was made using the SuperScript VILO Kit (Invitrogen).
1st PCR:  The cDNA is used as a template for a PCR reaction which pools all of the positive strand TCR Valpha oligos.   Each PCR reaction uses a Cα primer, negative strand (see Silberman & al. (2016), PNAS 113(38):E5608-17, doi: 10.1073/pnas.1609717113 for primer sequences).  The positive strand oligo is specific for 20 different Vα’s (some families require more than one oligo) and adds suitable adapters for either 454 or Ion Torrent sequencers. All PCR products are separated by size on a 1.5% TBE agarose gel and excised from the gel in the range of 250 bp- 500 bp. 2nd PCR: A reaction using 2.5 ul of the pooled bands is prepared.   The positive primer binds to the sequencing adapter.  The negative primer is just 5’ of the Cα oligo used in the first PCR and adds a bar code which denotes the cell sample used. Products are run on a 1.5% TBE gel and the wide band (500-700 bp) is excised from the gel and extracted using a Qiaquik kit without heating.

value: B6, MHCs, TCRb-/-, Tg DObWT+, TCRA+/-


title: MLF273

description: Strand-specific RNA-seq (dUTP)

value: Mixed C57BL/6-129

value: Cic(lox/lox) hUBC-CreERT2(+/T)


title: Control, SR


title: RNG105 cKO, SP

description:
cKO_SP-vs-SR_gene_exp.diff
SP_cont-vs-cKO_gene_exp.diff

source_name: Hippocampus, SP

value: hippocampal SP


title: MGAS10870 B

source_name: MGAS10870 B