title: LM2-H3K9me3


title: MDA231-Pol-II

status: Public on Jul 31 2019

type: SRA

city: Shanghai

first: Ji

last: Zhang

state: Select a State or Province

address: 197 Ruijin 2nd Road

country: China

institute: Shanghai Jiao Tong University School of Medicine

library_source: genomic

data_processing:
All of the reads were mapped to unmasked human reference genome (hg19) using Bowtie2 with default parameters
Only uniquely mapped reads were retained, and duplicated reads were compressed to single read by samtools
ChIP-seq peak calling was performed using MACS v2.0.10(32), with“–broad” option.
Regions with q-value less than 0.01 were identified as peaks
For each cell line, the respective ChIP inputs were used as control data
Genome_build: hg19
Supplementary_files_format_and_content: bedGraph files were generated using bedtools

molecule: genomic DNA

organism: Homo sapiens

source_name: MDA-MB-231 cell line

growth_protocol: The human breast cancer cell line MDA-MB-231 and LM2-4175 were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS (Gibco BRL, Grand Island, NY, USA) at 37°C and 5% CO2 in a humidified incubator

extract_protocol:
For chromatin immunoprecipitation, MDA-MB-231 and LM2-4175 cells were harvested and performed by ChIP-IT High Sensitivity kit (Active Motif, #53040) according to manufacturer's instructions
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303)

tag: tissue

value: breast cancer cell line

tag: cell line

tag: chip antibody

value: Pol-II

tag: antibody vendorname

value: Abcam

tag: antibody vendorid

tag: treatment


title: 1702

city: Chapel Hill

first: Karen

middle: L

last: Mohlke

state: NC

address: 120 Mason Farm Rd

department: Genetics

country: USA

institute: The University of North Carolina at Chapel Hill

library_source: transcriptomic

data_processing:
Fastx toolkit was used to remove low quality reads; reads with phred quality > 20 for 80% of bases were retained.
Tagdust was used to identify and remove reads containing adapter and linker sequences.
STAR (version 2.4.2a) was used as the aligner, with hg19 and GENCODE v19 as annotation; STAR was run, using a 2-pass strategy, were splice locations from the first pass were used as additional annotation for the second pass.
salmon (version 0.7.2) was used to estimate isoform abundance from aligned BAM files; GENCODE 19 was used as annotation. --gcBias option was used.
tximport (version 1.0.3) was used to collapse isoform abundance estimates from salmon to gene abuddance estimates
Genome_build: hg19
Supplementary_files_format_and_content: Abundance measurements generated using salmon and tximport; values represent the transcripts per million (TPM) estimate of gene abundance.

molecule: polyA RNA

source_name: subcutaneous adipose tissue

extract_protocol:
Subcutaneous adipose needle biopsies were obtained from near the umbilicus of men from the METSIM cohort. RNA was isolated using the QIAGEN miRNeasy kit.
Libraries were constructed using a polyA+ selection protocol (Illumina TruSeq RNA Sample Preparation Kit v2)

tag: Sex

value: male

tag: sequencing batch

value: batch 3

tag: transcript integrity number

tag: age

tag: bmi


title: MDA231-H3K27ac


title: 1674


title: MDA-MB-231 replicate 1

data_processing:
RNA-Seq reads were mapped to human reference genome (hg19) using Tophat2 with default parameters
FPKM value for each gene was calculated by cuffdiff
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each gene in MDA-MB-231 and LM2-4175 cell lines

molecule: total RNA

growth_protocol: The human breast cancer cell line MDA-MB-231 and LM2-4175 were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS (Gibco BRL, Grand Island, NY, USA) at 37°C and 5% CO2 in a humidified incubator.

extract_protocol:
Cellular RNA was isolated by Tri-Reagent using the manufacturer’s instructions. Qualified total RNA was further purified by RNeasy micro kit and RNase-Free DNase Set.
RNA sequencing libraries were prepared according to the IlluminaTruseq TM  RNA sample prep Kit


title: MDA231-input

value: input


title: MDA231-H3K9me3


title: MDA-MB-231 replicate 2


title: 1666


title: MDA231-H3K4me1


title: 1653


title: 1516


title: 1619


title: 1483


title: 1504


title: XXFb170: HDF_Patient_170

city: Biella

first: Paola

last: Ostano

address: via Malta 3

country: Italy

institute: Fondazione Edo ed Elvo Tempia

data_processing:
Single read was done on Illumina HiSeq 2500 sequencer at the Genomic Technologies Facility of the University of Lausanne (Lausanne, Switzerland). Reads were trimmed by using Trimmomatic (v0.22), followed by mapping to human hg19 reference genome using TopHat2 (v2.0.8b). Gene expression levels were then evaluated using HTSeq package (release 0.5.4p1).
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include log2 RPKM

source_name: Foreskin-derived fibroblasts

growth_protocol: Primary human fibroblasts derived from foreskins of circumcised 1-year-old to 5-year-old healthy males, cultured in complete medium (DMEM enriched in Glutamine, Pyruvate and high Glucose level supplemented with 10% FBS and 1% Antibiotic Cocktail)

extract_protocol:
RNA was extracted from HDFs through Direct-zol RNA MiniPrep kit (Zymo Research) with on-column DNase treatment and its quality was verified by a Bioanalyzer (Agilent Technologies)
1 ug of total RNA per sample was used for library preparation using a NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs).

value: Foreskin-derived fibroblasts


title: 1452


title: XXFb171: HDF_Patient_171


title: 1678


title: XXFb173: HDF_Patient_173


title: 1366


title: XXFb175: HDF_Patient_175


title: XXFb165: HDF_Patient_165


title: XXFb166: HDF_Patient_166


title: XXFb176: HDF_Patient_176


title: MDA231-H3K27me3


title: 1353


title: XXFb163: HDF_Patient_163


title: 1343


title: 1303


title: 1223


title: 1228


title: XXFb159: HDF_Patient_159


title: 1162


title: XXFb162: HDF_Patient_162


title: 1165


title: XXFb158: HDF_Patient_158


title: 1518


title: XXFb160: HDF_Patient_160


title: XXFb156: HDF_Patient_156


title: XXFb164: HDF_Patient_164


title: 1160


title: 1086


title: 844


title: 874


title: 908


title: 598


title: XXFb152: HDF_Patient_152


title: XXFb151: HDF_Patient_151


title: XXFb149: HDF_Patient_149


title: 575


title: 482


title: 6553


title: XXFb139: HDF_Patient_139


title: XXFb144: HDF_Patient_144


title: XXFb145: HDF_Patient_145


title: XXFb146: HDF_Patient_146


title: 1278


title: 7601


title: XXFb157: HDF_Patient_157


title: XXFb137: HDF_Patient_137


title: XXFb155: HDF_Patient_155


title: XXFb154: HDF_Patient_154


title: 1157


title: XXFb150: HDF_Patient_150


title: 951


title: XXFb136: HDF_Patient_136


title: 2974


title: XXFb135: HDF_Patient_135


title: 520


title: XXFb134: HDF_Patient_134


title: XXFb148: HDF_Patient_148


title: 697


title: XXFb133: HDF_Patient_133


title: myc2,3,4 vs Col-0 WL repl4

type: RNA

city: Madrid

first: Jose M.

last: Franco-Zorrilla

state: Madrid

address: Darwin, 3

department: Plant Molecular Genetics

country: Spain

institute: Centro Nacinal de Biotecnologia-CSIC

hyb_protocol: 825 ng of Cy3 and Hy5 probes were mixed and hybridized for 17 hours at 65ºC in GEx Hybridization Buffer HI-RPM in a hybridization oven. Arrays were washed according to the manufacturer’s instructions and dried by centrifugation.

scan_protocol: Images were scanned at 5um resolution on Agilent DNA Microarrays Scanner (G2565BA, Agilent Technologies) and analyzed with Feature Extraction software (Agilent Technologies).

data_processing: Raw intensities were background-substracted by NORMEXP method with a offset of 50. Signals (in log2 scale) were normalized by LOWESS algorithm (intra-arrays normalization) followed by adjustment of their quantiles (inter-arrays normalization).

organism: Arabidopsis thaliana

source_name: Arabidopsis thaliana Col-0

label_protocol: 200 ng of total RNA were reverse transcribed using oligo dT-T7 primer, and cDNA converted into aRNA using T7 RNA polymerase and incorporating cyanine 3- or Hyper 5-labeled CTP. All the step were carried put according to 'Two color Microarray-Based GeneExpression Analysis Protocol', Agilent Technologies )

growth_protocol: Seeds were stratified in darkness at 4°C for 3 d and grown in a 16/8-h photoperiod at 21°C in White Light (WL; PAR, 100 mmol m22 s21; R:FR ratio = 11.7) or White Light  supplemented with Far Red (FR; PAR, 100 micromol m-2 s-1, R:FR ratio = 0.2) for 3 hours.

extract_protocol: Total RNA was extracted using TRIzol reagent (Invitrogen, 15596-026) according to manufacturer's instructions and purified with an RNeasy mini kit (Qiagen, 74104).

tag: genotype

value: wild-type

value: White Light


title: 7246


title: 1408


title: XXFb132: HDF_Patient_132


title: XXFb118: HDF_Patient_118


title: 5009


title: 1517


title: 4509


title: XXFb112: HDF_Patient_112


title: XXFb108: HDF_Patient_108


title: 1524


title: XXFb107: HDF_Patient_107


title: 378


title: XXFb105: HDF_Patient_105


title: myc2,3,4 vs Col-0 WL repl2


title: 541


title: 6572


title: XXFb80: HDF_Patient_80


title: myc2,3,4 vs Col-0 FR repl2

value: Far Red Light


title: KLFb141: HDF_Patient_141


title: 6557


title: myc2,3,4 vs Col-0 WL repl1


title: myc2,3,4 vs Col-0 WL repl3


title: Aza1

city: Coleraine

first: Colum

middle: Patrick

last: Walsh

state: County Londonderry

address: Cromore Road

department: Life and Health Sciences

country: United Kingdom

institute: Ulster University

description: Aza treated hTERT replicate 2

hyb_protocol: Standard Illumina hybridization protocol.

scan_protocol: Standard Illumina scanning protocol.

data_processing:
Illumina proprietary software, GenomeStudio.
R using the lumi package and the limma package.

label: biotin

source_name: hTERT

label_protocol: Standard Illumina Protocol.

growth_protocol: The parental or wild type (WT) adherent hTERT1604 lung fibroblast cell line was cultured in 4.5g/l glucose DMEM supplemented with 10% FBS and 2x NEAA. Aza treated hTERT1604 cells were maintained as per WT.

extract_protocol:
Total RNA was extracted using the RNeasy minikit (Qiagen) as per the manufacturer’s instructions; a DNase step was included to ensure the removal of contaminating genomic DNA.
The concentration, purity and integrity of the resulting cRNA were measured by SpectroStar and bioanalyser.

treatment_protocol: Aza treatment was performed by seeding hTERT-1604 cells onto a 90 mm plate in medium (as per WT) and allowed to attach overnight before replacing with complete medium containing 1 ?M Aza, which was renewed at 24-hour intervals up to 72 hours.

tag: gender

value: Male


title: Aza2

description: Aza treated hTERT replicate 3


title: Aza

description: Aza treated hTERT replicate 1


title: KLFb140: HDF_Patient_140


title: myc2,3,4 vs Col-0 FR repl4


title: myc2,3,4 vs Col-0 FR repl3


title: KLFb138: HDF_Patient_138


title: 5606


title: 5538


title: 5845


title: KLFb130: HDF_Patient_130


title: myc2,3,4 vs Col-0 FR repl1


title: KLFb127: HDF_Patient_127


title: MDA231-H3K4me3


title: KLFb126: HDF_Patient_126


title: KLFb131: HDF_Patient_131


title: KLFb125: HDF_Patient_125


title: 4485


title: 5203


title: 4403


title: KLFb120: HDF_Patient_120


title: 6006


title: KLFb116: HDF_Patient_116


title: KLFb122: HDF_Patient_122


title: 2869


title: KLFb114: HDF_Patient_114


title: Phf6-KO2

city: New York

first: Stuart

middle: Aidan

last: Quinn

state: NY

address: 1130 St Nicholas Ave

department: Institute for Cancer Genetics

institute: Columbia University

data_processing:
Demultiplexing was performed and fastq files generated using bcl2fastq v1.8.4 (illumina)
Reads were aligned to GRCm38 using STAR v2.6.0a guided by splice junctions included in the comprehensive gene annotation set release M17 (Gencode)
Gene-level read counts were computed using featureCounts (Rsubread)
Counts were normalized and tested for differential expression under a negative binomial model in DESeq2 (R package)
Genome_build: GRCm38
Supplementary_files_format_and_content: DESeq2 normalized, gene-level counts

organism: Mus musculus

source_name: Isogenic T-ALL tumor harvested from bone marrow

extract_protocol:
Total RNA was extracted using Qiagen RNeasy Mini Kit (cat number 74004)
NEBNext Ultra RNA Library Prep Kit for Illumina was used for cDNA synthesis and library preparation per manufacturer’s specifications. Pooled libraries were sequenced on an Illumina HiSeq platform using a 2x150 Paired End (PE) configuration to generate approximately 58 million reads per sample.

treatment_protocol: Tamoxifen or vehicle only (corn oil) was administered by intraperitoneal injection to induce expression of the tamoxifen-inducible Cre recombinase resulting in conditional deletion of Phf6.

tag: strain


title: 2760


title: Phf6-WT2


title: Phf6-KO1


title: KLFb113: HDF_Patient_113


title: KLFb111: HDF_Patient_111


title: Phf6-WT1


title: 2692


title: Phf6-KO3


title: 2415


title: Ribo-seq_Intact PTEN RP RNA rep2

city: Seattle

first: Sonali

last: Arora

state: WA

address: 1100 Fairview Ave N,

institute: FHCRC

description: Intact PTEN RP RNA rep2

data_processing:
Library strategy: Ribo-Seq
Sequencing was performed using an Illumina HiSeq 2000 in Rapid Run mode and  employed a paired-end, 50 base read length (PE50) sequencing strategy.
All reads were aligned to the UCSC mm10 assembly using Tophat2 and counted for gene associations against the UCSC genes database with HTSeq
All data was combined and normalized using a CPM method from the R/Bioconductor package, edgeR
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include cpm values for each Sample.

source_name: prostate

extract_protocol:
RNA was extracted from cell cultures every two weeks during the exponential growth phase; RNA was isolated from 0.5-1.0×10^6 cells. Total RNA was prepared using RNeasy Mini Kit with DNase I (Qiagen). RNA concentration and quality (A260/A280) was measured using a NanoDrop 1000 Spectrophotometer (Thermo Scientific)
RNA libraries were prepared for sequencing using standard Illumina protocols

value: prostate

tag: castration status

value: Intact

tag: rna fraction

value: RP RNA


title: 4951


title: 2687


title: Ribo-seq_Intact PTEN RP RNA rep1

description: Intact PTEN RP RNA rep1


title: Phf6-WT3


title: KLFb109: HDF_Patient_109


title: Ribo-seq_Intact PTEN Total RNA rep2

description: Intact PTEN Total RNA rep2

value: Total RNA


title: KLFb110: HDF_Patient_110


title: Ribo-seq_Castrate PTEN RP RNA rep3

description: Castrate PTEN RP RNA rep3

value: Castrate


title: Ribo-seq_Castrate PTEN Total RNA rep3

description: Castrate PTEN Total RNA rep3


title: KLFb106: HDF_Patient_106


title: Ribo-seq_Intact PTEN Total RNA rep1

description: Intact PTEN Total RNA rep1


title: 2044


title: Ribo-seq_Castrate PTEN RP RNA rep2

description: Castrate PTEN RP RNA rep2


title: 1784


title: KLFb103: HDF_Patient_103


title: Ribo-seq_Castrate PTEN Total RNA rep2

description: Castrate PTEN Total RNA rep2


title: 1167


title: 1111


title: 817


title: 7552


title: 5381


title: SRSF2-KO liver

first: cheng

middle: yuan

last: ming

address: NO.320 Yueyang Road

institute: Shanghai Institutes for Biological Science CAS

data_processing:
Illumina GA Pipeline v1.3 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm9 whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
The stat of mapped reads was list in WT.gene.MapReadsStat.xls and KO.gene.MapReadsStat.xls
Reads Per Kilobase of exon per Megabase of library size (FRPKM) were calculated using a protocol from Mortazavi, 2008.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample

source_name: liver from mice at 5 month after born

extract_protocol:
liver at 5 month after born were collected and frozened in Liquid nitrogen, and RNA was harvested using Trizol reagent.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: BALB/c

value: 5 month after born

value: liver


title: KLFb101: HDF_Patient_101


title: RNA-seq_Intact Replicate 1

data_processing:
Sequencing was performed using an Illumina HiSeq 2000 in Rapid Run mode and  employed a paired-end, 50 base read length (PE50) sequencing strategy.
RNA-seq reads were aligned to the UCSC mm10 assembly using Tophat2 and counted for gene associations against the UCSC genes database with HTSeq
All data was combined and normalized using a CPM method from the R/Bioconductor package, edgeR
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include cpm values for each Sample.

extract_protocol:
Total RNA was prepared using RNeasy Mini Kit with DNase I (Qiagen). RNA concentration and quality (A260/A280) was measured using a NanoDrop 1000 Spectrophotometer (Thermo Scientific)
RNA libraries were prepared for sequencing using standard Illumina protocols


title: WT liver

value: WT


title: KLFb99: HDF_Patient_99


title: 906

value: male


title: 490

value: batch 3


title: RNA-seq_Intact Replicate 2


title: RNA-seq_Cx Replicate 4

value: Cx


title: KLFb97: HDF_Patient_97

value: Foreskin-derived fibroblasts


title: 904


title: PA3880-1-anaerobic [PA3880-PAK1900-1-anaerobic]

city: Hong Kong

first: Xin

last: DENG

address: 1/F, Block 1, To Yuen Building, City University of Hong Kong, 31 To Yuen Street, Kowloon Tong

department: Department of Biomedical Sciences

country: Hong Kong

institute: City University of Hong Kong

data_processing:
Bacterial RNA-seq reads were mapped to the P. auruginosa genome by using TopHat (version 2.0.0), with two mismatches allowed
Only the uniquely mapped reads were kept for the subsequent analyses.
The gene differential expression analysis was performed using Cuffdiff software (version 2.0.0)
Genome_build: Pseudomonas aeruginosa PAO1
Supplementary_files_format_and_content: xls file containing comparisons between AnvM mutant and wild-type

organism: Pseudomonas aeruginosa PAO1

source_name: anvM mutant_mid-log phase in LB under the anaerobic

growth_protocol: liquid culture in LB (Luria-Bertani)  medium

extract_protocol:
To examine the effect of AnvM on the transcriptome, under the aerobic and anaerobic conditions 2 ml of mid-log-phase (OD600= 0.6) bacterial cultures (PAO1 and ∆anvM strains) were collected by centrifugation (12,000 rpm, 4℃). An RNeasy minikit (Qiagen) was used for subsequent RNA purification with DNaseI (NEB) treatment. After removing rRNA by using the MICROBExpress kit (Ambion), mRNA was used to generate the cDNA library according to the TruSeq RNA sample prep kit protocol (Illumina), which was then sequenced using the HiSeq 2000 system (Illumina). Bacterial RNA-seq reads were mapped to the P. auruginosa genome (ASM676v1, NC 002516.2) by using TopHat (version 2.0.0), with two mismatches allowed. Only the uniquely mapped reads were kept for the subsequent analyses. The gene differential expression analysis was performed using Cuffdiff software (version 2.0.0). GO enrich-ment analyses were conducted on all differentially transcribed genes using DAVID before data sets were imported into Cytoscape with an Enrichment Map plugin. The each sample in RNA-seq assay was repeated twice.
TruSeq RNA sample prep kit protocol (Illumina)

treatment_protocol: grow until mid-log phase in LB under the anaerobic

tag: genotype/variation

value: anvM mutant

tag: growth condition

value: mid-log phase in LB under the anaerobic


title: Ribo-seq_Castrate PTEN RP RNA rep1

description: Castrate PTEN RP RNA rep1

tag: tissue


title: PA3880-2-anaerobic [PA3880-PAK1900-2-anaerobic]


title: KLFb95: HDF_Patient_95


title: mm_Egr1_ChIP-seq rep1+rep2

city: Bethesda

first: Mingan

last: Sun

state: Maryland

address: Building 6B, 6 Center Drive

department: Eunice Kennedy Shriver National Institute of Child Health and Human Development

institute: National Institute of Health

data_processing:
Sequencing adapters in raw sequencing reads were removed by Cutadapt (v.11.2). Moreover, the bad quality of bases in either ends of reads were trimmed by a customized Perl script. After trimming, reads were mapped to mm10 by Bowtie2 (v2.2.9). Subsequently, SPP (v1.13) was used to call narrow peaks on two replicates and IDR (v2.0.2) was used to evaluate the reproducibility of pairwise replicates. Significant and reproducible peaks on two replicates were reported.  In Bedtools (v2.26.0), the subroutine "genomeCov" was used to generate visualized signals (BEDGRAPH format), which were normalized to 1 million (Reads per million, RPM).
Genome_build: mm10
Supplementary_files_format_and_content: BEDGRAPH

source_name: Frontal cortex

extract_protocol: ChIP-seq library:      129SvEv/C57BL/6N F1 hybrid male mice were produced from breeding trios of one C57BL/6N male and two 129SvEv females. Mice were anesthetized with avertin and decapitated. Brains were chilled in ice-cold phosphate buffer saline and sliced using a Vibratome (Ted Pella) into 300 mm slices. Frontal cortices were micro-dissected, minced into small pieces (~1 mm3) and immediately cross-linked in 1% formaldehyde for 20 min at room temperature (RT), and quenched by adding 125 mM glycine for 5 min at RT. The tissue was then rinsed twice in cold phosphate-buffered saline (PBS) containing halt protease inhibitor cocktail (Pierce Biotechnology, Inc.) and centrifuged at 850 g for 10 min at 4 °C. Tissue pellets were homogenized in lysis buffer. After clarifying the lysate from sonicated nuclei, the genomic DNA fragments were obtained from anti-EGR1 immunoprecipitated chromatin. Libraries were prepared according to the protocol developed by Blecher-Gonen et al. (PMID: 23429716)

value: brain

value: 6 week


title: 10886

value: batch 2


title: RNA-seq_Cx Replicate 3

value: prostate


title: RNA-seq_Cx Replicate 1


title: Ribo-seq_Castrate PTEN Total RNA rep1

description: Castrate PTEN Total RNA rep1


title: RNA-seq_Cx Replicate 2


title: KLFb98: HDF_Patient_98


title: KLFb96: HDF_Patient_96


title: PAO1-2-anaerobic [PA01-PAK1900-2-anaerobic]

source_name: WT_mid-log phase in LB under the anaerobic


title: KLFb79: HDF_Patient_79


title: 10392


title: PAO1-1-aerobic [PA01-PAK1900-1]

source_name: WT_mid-log phase in LB under the aerobic

treatment_protocol: grow until mid-log phase in LB under the aerobic

value: mid-log phase in LB under the aerobic


title: KLFb78: HDF_Patient_78


title: PA3880-1-aerobic [PA3880-PAK1900-1]

source_name: anvM mutant_mid-log phase in LB under the aerobic


title: 10805


title: mm_input_DNA

extract_protocol: ChIP-seq library:      129SvEv/C57BL/6N F1 hybrid male mice were produced from breeding trios of one C57BL/6N male and two 129SvEv females. Mice were anesthetized with avertin and decapitated. Brains were chilled in ice-cold phosphate buffer saline and sliced using a Vibratome (Ted Pella) into 300 mm slices. Frontal cortices were micro-dissected, minced into small pieces (~1 mm3) and immediately cross-linked in 1% formaldehyde for 20 min at room temperature (RT), and quenched by adding 125 mM glycine for 5 min at RT. The tissue was then rinsed twice in cold phosphate-buffered saline (PBS) containing halt protease inhibitor cocktail (Pierce Biotechnology, Inc.) and centrifuged at 850 g for 10 min at 4 °C. Tissue pellets were homogenized in lysis buffer. After clarifying the lysate from sonicated nuclei, the genomic DNA fragments were obtained from anti-EGR1 immunoprecipitated chromatin.Libraries were prepared according to the protocol developed by Blecher-Gonen et al. (PMID: 23429716)


title: 096_SCT80_AIT_015_Bcells_H12

city: La Jolla

first: Veronique

last: Shulten

state: CA

address: 9420 Athena Circle

institute: LJI

data_processing:
Reads passing Illumina filters were further filtered for those mapping to tRNA, rRNA, adapters, and spike-in controls using Bowtie v1.0.0 (-S --fr)
Filtered reads were mapped against the mm9 reference using Tophat v1.4.1 (--library-type fr-firststrand)
Reads mapping to the reference genome are further filtered to remove low-complexity reads with PRINSEQ v0.20.3 (-lc_method "dust" -lc_threshold 4)
Sequencing read coverage per gene was counted using HTSeq-count v0.7.1 (-m union -s yes -t exon -i gene_id) and features with 0 counts across all samples were removed
Samples were then processed in R using the Seurat package
Genome_build: hg19
Supplementary_files_format_and_content: read count matrix with rows as genes and columns as cells

source_name: B cell single cell seq

growth_protocol: unstimulated B cells in RPMI medium with 5% human serum

extract_protocol:
Direct sorting into lysis buffer
This assay was adapted from the Smart-seq2 protocol (Picelli et al. 2013, Rosales et al. 2018). Single cells were sorted into wells of 96-well PCR plates containing 4 μl cell lysis buffer composed by 0.2% Triton X-100, 2 U/ul of recombinant RNase inhibitor (Clontech/Takara), 5 mM dNTP mix (Life Technologies). The sort stream was calibrated carefully to ensure that the cells landed in the liquid interface. RNA loss was minimized by performing on-plate RNA reverse-transcription and whole transcriptome amplification to generate ~1–10 ng of cDNA. Briefly, mRNA was captured using poly-dT oligos and directly reverse-transcribed into full-length double stranded cDNA using the described template-switching LNA oligo (Picelli et al. 2013). Whole transcriptome cDNA was amplified by PCR  and purified using AMPure XP magnetic bead solution (0.9:1 (vol:vol) ratio, Beckman Coulter) twice. The number of PCR cycles for optimal cDNA amplification was determined by qPCR. Quality and quantity of cDNA amplification were assessed by Fragment Analyzer (Advance Analytical) and fluorescent dsDNA intercalating-dye based assay (Picogreen, Invitrogen). Samples that failed quality control steps as described (Rosales et al.2018) were eliminated from downstream steps. Standard qPCR was performed for housekeeping genes to ensure comparable amplification of all single-cell samples. 0.4 ng of cDNA was used to prepare a standard Nextera XT sequencing library (NextEra XT DNA library prep kit and index kits; Illumina), using an automated platform (Biomek FXp, Beckman Coulter). Before sequencing all libraries were purified using AMPure XP beads and quality control steps performed as listed previously. Samples that passed strict quality controls were pooled at equimolar concentration, loaded on flow cells and sequenced on the Illumina Sequencing platform, HiSeq2500 (Illumina). Libraries were sequenced to obtain more than 0.3 million 150-bp pair-end reads (HiSeq Rapid Run PE Cluster and SBS Kit V2; Illumina) mapping uniquely to mRNA reference.
single cell RNAseq based on Smart  seq 2

treatment_protocol: ex vivo staining and FACS sorted

value: blood

tag: cell type

value: peripheral blood mononuclear cells

tag: cell subtype

value: B cell

tag: true ige producing memory b cells

value: no


title: 10704


title: 10738


title: PA3880-2-aerobic [PA3880-PAK1900-2]


title: PAO1-2-aerobic [PA01-PAK1900-2]


title: 095_SCT80_AIT_015_Bcells_G12


title: 094_SCT80_AIT_015_Bcells_F12


title: 093_SCT80_AIT_015_Bcells_E12


title: 10752


title: 092_SCT80_AIT_015_Bcells_D12


title: PAO1-1-anaerobic [PA01-PAK1900-1-anaerobic]


title: 090_SCT80_AIT_015_Bcells_B12


title: 10565


title: 10016


title: 10548


title: 089_SCT80_AIT_015_Bcells_A12


title: 088_SCT80_AIT_015_Bcells_H11


title: 10790


title: 10437


title: 087_SCT80_AIT_015_Bcells_G11


title: 10425


title: 10390


title: 085_SCT80_AIT_015_Bcells_E11


title: 10688


title: 086_SCT80_AIT_015_Bcells_F11


title: 10567


title: 10300


title: 081_SCT80_AIT_015_Bcells_A11


title: 080_SCT80_AIT_015_Bcells_H10


title: 079_SCT80_AIT_015_Bcells_G10


title: 091_SCT80_AIT_015_Bcells_C12


title: 10039


title: 10096


title: 10012


title: 078_SCT80_AIT_015_Bcells_F10


title: Expression Sorted  CT Library; Replicate 2

city: Urbana

first: Erik

last: Procko

state: IL

address: 601 S Goodwin Ave

department: Biochemistry

institute: University of Illinois

description: Deep mutational scan of SERT for expression

data_processing:
Data was analyzed using Enrich: http://depts.washington.edu/sfields/software/enrich/
Fuser Script: Fragmnt NT A read1: python2.7 Paired_read_fuser.py --path NT_fragA_R1/ --read1 SERT_SCIT_R1.fastq.bz2 --read2 SERT_SCIT_R1.fastq.bz2 --wtseq GAGACCACCCCCCTGAACAGTCAAAAGCAACTCTCTGCCTGTGAGGATGGAGAGGACTGTCAGGAGAATGGCGTTCTTCAAAAGGTTGTACCCACCCCTGGAGATAAGGTGGAAAGCGGCCAGATTTCCAACGGCTACAGCGCCGTGCCATCACCTGGAGCCGGGGATGATACACGGCACAGCATTCCCGCAACCACCACGACACTG --read1_overlap_start 29 --read1_overlap_end 236 --mode R1
Fuser_library_NT_fragA_R2.sh: python2.7 Paired_read_fuser.py --path NT_fragA_R2/ --read1 SERT_SCIT_R2.fastq.bz2 --read2 SERT_SCIT_R2.fastq.bz2 --wtseq TTCAAGGGAATCGGATACGCAATCTGTATAATCGCGTTCTATATTGCATCATATTACAACACCATCATGGCCTGGGCCCTTTATTACTTGATCAGCAGTTTCACCGACCAATTGCCGTGGACGAGCTGTAAGAACTCATGGAATACCGGGAACTGTACCAACTACTTCTCTGAGGACAGCTCAGGAGGTAGCGGGGGTGAACAG --read2_overlap_start 23 --read2_overlap_end 227 --mode R2
Fuser_library_NT_fragB_R1.sh: python2.7 Paired_read_fuser.py --path NT_fragB_R1/ --read1 SERT_SCIT_R1.fastq.bz2 --read2 SERT_SCIT_R1.fastq.bz2 --wtseq GCAACCACCACGACACTGGTTGCAGAGCTTCACCAGGGCGAGAGGGAGACGTGGGGCAAAAAGGTAGATTTTCTGCTTTCCGTGATCGGTTACGCGGTCGATCTGGGGAACGTGTGGAGGTTCCCTTACATTTGTTACCAAAACGGAGGGGGTGCCTTCTTGCTGCCCTACACAATTATGGCGATTTTTGGGGGTATCCCGCTCTTCTACATGG  --read1_overlap_start 21 --read1_overlap_end 235 --mode R1
Fuser_library_NT_fragB_R2.sh: python2.7 Paired_read_fuser.py --path NT_fragB_R2/ --read1 SERT_SCIT_R2.fastq.bz2 --read2 SERT_SCIT_R2.fastq.bz2 --wtseq CAGAAGCTGATTAGCGAGGAGGACTTGGGCAGTCCAGGCGGAAGCGGCAACATCACTTGGACACTCCATAGTACGAGCCCCGCAGAGGAGTTTTACACCAGGCACGTACTGCAGATTCATAGGAGCAAGGGTCTGCAAGACCTGGGCGGTATTAGCTGGCAGCTCGCATTGTGCATAATGTTGATCTTTACGGTGATCTATTTCTCT --read2_overlap_start 20 --read2_overlap_end 227 --mode R2
Fuser_library_NT_fragC_R1.sh: python2.7 Paired_read_fuser.py --path NT_fragC_R1/ --read1 SERT_SCIT_R1.fastq.bz2 --read2 SERT_SCIT_R1.fastq.bz2 --wtseq GAACTGGCCTTGGGTCAGTACCACCGGAACGGCTGCATTAGCATTTGGCGCAAGATATGCCCCATTTTCAAGGGAATCGGATACGCAATCTGTATAATCGCGTTCTATATTGCATCATATTACAACACCATCATGGCCTGGGCCCTTTATTACTTGATCAGCAGTTTCACCGACCAATTGCCGTGGACGAGCTGTAAG --read1_overlap_start 20 --read1_overlap_end 218 --mode R1
Fuser_library_NT_fragC_R2.sh: python2.7 Paired_read_fuser.py --path NT_fragC_R2/ --read1 SERT_SCIT_R2.fastq.bz2 --read2 SERT_SCIT_R2.fastq.bz2 --wtseq TTTACGGTGATCTATTTCTCTATCTGGAAGGGAGTAAAGACATCCGGCAAGGTGGTGTGGGTGACGGCGACCTTCCCCTACATTATACTCAGCGTGTTGTTGGTGAGGGGGGCAACCCTGCCTGGGGCCTGGCGAGGCGTGCTGTTCTATCTCAAGCCAAACTGGCAGAAGCTTCTTGAAACCGGAGTGTGGATCGACGCG --read2_overlap_start 28 --read2_overlap_end 229 --mode R2
Fuser_library_CT_fragA_R1.sh: python2.7 Paired_read_fuser.py --path CT_fragA_R1/ --read1 SERT_CT_DNA1_R1.fastq.bz2 --read2 SERT_CT_DNA1_R1.fastq.bz2 --wtseq TGGATCGACGCGGCCGCGCAAATCTTCTTTTCCCTCGGTCCCGGCTTCGGGGTCCTTCTGGCATTCGCAAGCTACAACAAGTTCAACAATAACTGCTACCAGGACGCCCTCGTGACAAGTGTAGTGAACTGCATGACCTCTTTCGTGAGCGGGTTTGTGATCTTCACAGTACTCGGATATATGGCTGAGATGAGGAACGAAGAT --read1_overlap_start 26 --read1_overlap_end 230 --mode R1
Fuser_library_CT_fragA_R2.sh: python2.7 Paired_read_fuser.py --path CT_fragA_R2/ --read1 SERT_CT_DNA1_R2.fastq.bz2 --read2 SERT_CT_DNA1_R2.fastq.bz2 --wtseq CTGGATAGCACCTTCGCCGGACTGGAGGGTGTCATCACTGCCGTTCTCGACGAGTTCCCCCATGTGTGGGCCAAGCGACGGGAAAGGTTCGTGCTGGCCGTCGTAATCACATGCTTCTTCGGCTCACTGGTGACCCTTACCTTTGGCGGTGCGTACGTGGTCAAGCTGCTCGAAGAGTACGCCACTGGACCGGCG --read2_overlap_start 35 --read2_overlap_end 230 --mode R2
Fuser_library_CT_fragB_R1.sh: python2.7 Paired_read_fuser.py --path CT_fragB_R1/ --read1 SERT_CT_DNA1_R1.fastq.bz2 --read2 SERT_CT_DNA1_R1.fastq.bz2 --wtseq GTTAGTGAGGTTGCCAAAGACGCTGGCCCGAGCCTGTTGTTCATCACATACGCCGAGGCTATCGCGAACATGCCCGCATCTACTTTCTTCGCCATCATCTTTTTCCTGATGCTGATAACCCTTGGGCTGGATAGCACCTTCGCCGGACTGGAGGGTGTCATCACTGCCGTTCTCGACGAGTTCCCCCATGTGTGGGCCAAG --read1_overlap_start 21 --read1_overlap_end 222 --mode R1
Fuser_library_CT_fragB_R2.sh: python2.7 Paired_read_fuser.py --path CT_fragB_R2/ --read1 SERT_CT_DNA1_R2.fastq.bz2 --read2 SERT_CT_DNA1_R2.fastq.bz2 --wtseq GTTCTGACCGTGGCATTGATCGAGGCCGTGGCCGTAAGCTGGTTTTACGGCATCACGCAGTTCTGTAGGGACGTGAAAGAGATGCTGGGCTTTTCTCCCGGTTGGTTTTGGAGGATTTGTTGGGTAGCAATTAGCCCCCTGTTTCTCCTTTTCATTATTTGCTCTTTTCTGATGTCTCCACCGCAGCTCCGACTGTTTCAGTACAACTACCCC --read2_overlap_start 15 --read2_overlap_end 228 --mode R2
Fuser_library_CT_fragC_R1.sh: python2.7 Paired_read_fuser.py --path CT_fragC_R1/ --read1 SERT_CT_DNA1_R1.fastq.bz2 --read2 SERT_CT_DNA1_R1.fastq.bz2 --wtseq CTGGATAGCACCTTCGCCGGACTGGAGGGTGTCATCACTGCCGTTCTCGACGAGTTCCCCCATGTGTGGGCCAAGCGACGGGAAAGGTTCGTGCTGGCCGTCGTAATCACATGCTTCTTCGGCTCACTGGTGACCCTTACCTTTGGCGGTGCGTACGTGGTCAAGCTGCTCGAAGAGTACGCCACTGGACCGGCG --read1_overlap_start 19 --read1_overlap_end 214 --mode R1
Fuser_library_CT_fragC_R2.sh: python2.7 Paired_read_fuser.py --path CT_fragC_R2/ --read1 SERT_CT_DNA1_R2.fastq.bz2 --read2 SERT_CT_DNA1_R2.fastq.bz2 --wtseq CCCTACTGGTCAATTATCCTGGGCTATTGTATCGGCACCTCAAGTTTCATCTGCATCCCCACATACATCGCATACAGACTGATAATTACGCCCGGTACTTTTAAGGAGCGAATCATTAAAAGCATAACCCCCGAAACCCCCACCGAGATCCCTTGTGGCGATATAAGACTCAATGCGGTC --read2_overlap_start 30 --read2_overlap_end 210 --mode R2
Aligner Script: Fragment NT_A Read 1: python2.7 Fused_read_aligner.py --path NT_fragA_R1/ --infile SERT_library_NT_R1.fast_R1_qc1 --referenceDNA GAGACCACCCCCCTGAACAGTCAAAAGCAACTCTCTGCCTGTGAGGATGGAGAGGACTGTCAGGAGAATGGCGTTCTTCAAAAGGTTGTACCCACCCCTGGAGATAAGGTGGAAAGCGGCCAGATTTCCAACGGCTACAGCGCCGTGCCATCACCTGGAGCCGGGGATGATACACGGCACAGCATTCCCGCA --referenceAA ETTPLNSQKQLSACEDGEDCQENGVLQKVVPTPGDKVESGQISNGYSAVPSPGAGDDTRHSIPA --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner Script: Fragment NT_A Read 2: python2.7 Fused_read_aligner.py --path NT_fragA_R2/ --infile SERT_library_NT_R2.fast_R2_qc1 --referenceDNA TTCAAGGGAATCGGATACGCAATCTGTATAATCGCGTTCTATATTGCATCATATTACAACACCATCATGGCCTGGGCCCTTTATTACTTGATCAGCAGTTTCACCGACCAATTGCCGTGGACGAGCTGTAAGAACTCATGGAATACCGGGAACTGTACCAACTACTTCTCTGAGGACAGCTCAGGAGGTAGCGGGGGTGAACAG --referenceAA FKGIGYAICIIAFYIASYYNTIMAWALYYLISSFTDQLPWTSCKNSWNTGNCTNYFSEDSSGGSGGEQ --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner Script: Fragment NT_B Read 1: python2.7 Fused_read_aligner.py --path NT_fragB_R1/ --infile SERT_library_NT_R1.fast_R1_qc1 --referenceDNA GCAACCACCACGACACTGGTTGCAGAGCTTCACCAGGGCGAGAGGGAGACGTGGGGCAAAAAGGTAGATTTTCTGCTTTCCGTGATCGGTTACGCGGTCGATCTGGGGAACGTGTGGAGGTTCCCTTACATTTGTTACCAAAACGGAGGGGGTGCCTTCTTGCTGCCCTACACAATTATGGCGATTTTTGGGGGTATCCCGCTCTTCTACATGG --referenceAA ATTTTLVAELHQGERETWGKKVDFLLSVIGYAVDLGNVWRFPYICYQNGGGAFLLPYTIMAIFGGIPLFYM --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner Script: Fragment NT_B Read 2: python2.7 Fused_read_aligner.py --path NT_fragB_R2/ --infile SERT_library_NT_R2.fast_R2_qc1 --referenceDNA CAGAAGCTGATTAGCGAGGAGGACTTGGGCAGTCCAGGCGGAAGCGGCAACATCACTTGGACACTCCATAGTACGAGCCCCGCAGAGGAGTTTTACACCAGGCACGTACTGCAGATTCATAGGAGCAAGGGTCTGCAAGACCTGGGCGGTATTAGCTGGCAGCTCGCATTGTGCATAATGTTGATCTTTACGGTGATCTATTTCTCT --referenceAA QKLISEEDLGSPGGSGNITWTLHSTSPAEEFYTRHVLQIHRSKGLQDLGGISWQLALCIMLIFTVIYFS --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner Script: Fragment NT_C Read 1: python2.7 Fused_read_aligner.py --path NT_fragC_R1/ --infile SERT_library_NT_R1.fast_R1_qc1 --referenceDNA GAACTGGCCTTGGGTCAGTACCACCGGAACGGCTGCATTAGCATTTGGCGCAAGATATGCCCCATTTTCAAGGGAATCGGATACGCAATCTGTATAATCGCGTTCTATATTGCATCATATTACAACACCATCATGGCCTGGGCCCTTTATTACTTGATCAGCAGTTTCACCGACCAATTGCCGTGGACGAGCTGTAAG --referenceAA ELALGQYHRNGCISIWRKICPIFKGIGYAICIIAFYIASYYNTIMAWALYYLISSFTDQLPWTSCK --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner Script: Fragment NT_C Read 2: python2.7 Fused_read_aligner.py --path NT_fragC_R2/ --infile SERT_library_NT_R2.fast_R2_qc1 --referenceDNA TTTACGGTGATCTATTTCTCTATCTGGAAGGGAGTAAAGACATCCGGCAAGGTGGTGTGGGTGACGGCGACCTTCCCCTACATTATACTCAGCGTGTTGTTGGTGAGGGGGGCAACCCTGCCTGGGGCCTGGCGAGGCGTGCTGTTCTATCTCAAGCCAAACTGGCAGAAGCTTCTTGAAACCGGAGTGTGGATCGACGCG --referenceAA FTVIYFSIWKGVKTSGKVVWVTATFPYIILSVLLVRGATLPGAWRGVLFYLKPNWQKLLETGVWIDA --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner Script: Fragment CT_A read 1:  python2.7 Fused_read_aligner.py --path CT_fragA_R1/ --infile SERT_CT_DNA1_R1.fast_R1_qc1 --referenceDNA TGGATCGACGCGGCCGCGCAAATCTTCTTTTCCCTCGGTCCCGGCTTCGGGGTCCTTCTGGCATTCGCAAGCTACAACAAGTTCAACAATAACTGCTACCAGGACGCCCTCGTGACAAGTGTAGTGAACTGCATGACCTCTTTCGTGAGCGGGTTTGTGATCTTCACAGTACTCGGATATATGGCTGAGATGAGGAACGAAGAT --referenceAA WIDAAAQIFFSLGPGFGVLLAFASYNKFNNNCYQDALVTSVVNCMTSFVSGFVIFTVLGYMAEMRNED --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner Script: Fragment CT_A read 2: python2.7 Fused_read_aligner.py --path CT_fragA_R2/ --infile SERT_CT_DNA1_R2.fast_R2_qc1 --referenceDNA CTGGATAGCACCTTCGCCGGACTGGAGGGTGTCATCACTGCCGTTCTCGACGAGTTCCCCCATGTGTGGGCCAAGCGACGGGAAAGGTTCGTGCTGGCCGTCGTAATCACATGCTTCTTCGGCTCACTGGTGACCCTTACCTTTGGCGGTGCGTACGTGGTCAAGCTGCTCGAAGAGTACGCCACTGGACCGGCG --referenceAA LDSTFAGLEGVITAVLDEFPHVWAKRRERFVLAVVITCFFGSLVTLTFGGAYVVKLLEEYATGPA --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R2
Aligner Script: Fragment CT_B read 1: python2.7 Fused_read_aligner.py --path CT_fragB_R1/ --infile SERT_CT_DNA1_R1.fast_R1_qc1 --referenceDNA GTTAGTGAGGTTGCCAAAGACGCTGGCCCGAGCCTGTTGTTCATCACATACGCCGAGGCTATCGCGAACATGCCCGCATCTACTTTCTTCGCCATCATCTTTTTCCTGATGCTGATAACCCTTGGGCTGGATAGCACCTTCGCCGGACTGGAGGGTGTCATCACTGCCGTTCTCGACGAGTTCCCCCATGTGTGGGCCAA --referenceAA VSEVAKDAGPSLLFITYAEAIANMPASTFFAIIFFLMLITLGLDSTFAGLEGVITAVLDEFPHVWA --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner Script: Fragment CT_B read 2: python2.7 Fused_read_aligner.py --path CT_fragB_R2/ --infile SERT_CT_DNA1_R2.fast_R2_qc1 --referenceDNA GTTCTGACCGTGGCATTGATCGAGGCCGTGGCCGTAAGCTGGTTTTACGGCATCACGCAGTTCTGTAGGGACGTGAAAGAGATGCTGGGCTTTTCTCCCGGTTGGTTTTGGAGGATTTGTTGGGTAGCAATTAGCCCCCTGTTTCTCCTTTTCATTATTTGCTCTTTTCTGATGTCTCCACCGCAGCTCCGACTGTTTCAGTACAACTACCCC --referenceAA VLTVALIEAVAVSWFYGITQFCRDVKEMLGFSPGWFWRICWVAISPLFLLFIICSFLMSPPQLRLFQYNYP --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R2
Aligner Script: Fragment CT_C read 1: python2.7 Fused_read_aligner.py --path CT_fragC_R1/ --infile SERT_CT_DNA1_R1.fast_R1_qc1 --referenceDNA CTGGATAGCACCTTCGCCGGACTGGAGGGTGTCATCACTGCCGTTCTCGACGAGTTCCCCCATGTGTGGGCCAAGCGACGGGAAAGGTTCGTGCTGGCCGTCGTAATCACATGCTTCTTCGGCTCACTGGTGACCCTTACCTTTGGCGGTGCGTACGTGGTCAAGCTGCTCGAAGAGTACGCCACTGGACCGGCG --referenceAA LDSTFAGLEGVITAVLDEFPHVWAKRRERFVLAVVITCFFGSLVTLTFGGAYVVKLLEEYATGPA --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner Script: Fragment CT_C read 2:  python2.7 Fused_read_aligner.py --path CT_fragC_R2/ --infile SERT_CT_DNA1_R2.fast_R2_qc1 --referenceDNA CCCTACTGGTCAATTATCCTGGGCTATTGTATCGGCACCTCAAGTTTCATCTGCATCCCCACATACATCGCATACAGACTGATAATTACGCCCGGTACTTTTAAGGAGCGAATCATTAAAAGCATAACCCCCGAAACCCCCACCGAGATCCCTTGTGGCGATATAAGACTCAATGCGGTC --referenceAA PYWSIILGYCIGTSSFICIPTYIAYRLIITPGTFKERIIKSITPETPTEIPCGDIRLNAV --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R2
MapCounts script: Example: python mapCounts.py --path directory_fragA1_R1/ --infile SERT_NTlibrary_R1.fast_R1_qc1_PRO_qc2
MapRatios script: Example: python mapRatios.py --path ratios_directory/ --templatepath /deepseq_scripts/r_deepseq_scripts/ --infile2 mapcounts_library --infile1 mapcounts_sorted
MapParts script: Example: python mapParts.py --path ratios_directory/ --infile mapratios_sorted_library --mode mutations:1
Unlink script: Example: python mapUnlink.py --path ratios/ --infile mapratios_sorted_library.m1 --type protein --mode ratios --size ##
In Excel, the log(base2) enrichment ratio of the wildtype sequence was subtracted from the log(2) enrichment ratios for all single mutations.
Data was then assembled from the different fragments/reads to span the full sequence in Excel.  Where there was overlap, real enrichment ratios were averaged and converted back to log(base2). Maximum depletion was set to log(base2) enrichment ratio = -4.5.
Genome_build: Not applicable
Supplementary_files_format_and_content: Excel spreadsheet of log(base2) enrichment ratios for each single amino acid substitution.  Also includes the frequency of each mutation in the naïve plasmid library.

source_name: Synthetic coding sequences

growth_protocol: Expi293F cells cultured in Expi293 Expression Medium (Life Technologies)

extract_protocol:
Total RNA was harvested from sorted cells using GeneJet RNA Purification Kit (Thermo Scientific) and cDNA prepared using Accuscript Hi-Fi (Agilent Genomics) primed with a gene-specific primer: SERT_NT__rev (__________, NT Library) and EBV_reverse (GTGGTTTGTCCAAACTCATC, CT Library).
In the first round of PCR (20 cycles), the SERT coding sequences were amplified as overlapping fragments of ~700 bp each in the case of the NT Library or ~600bp for the CT library.
Primer pair (NT Library Fragment 1): MiSeq_pCEP4intron_F2 (5`TCTTTCCCTACACGACGCTCTTCCGATCTACGACTCACTATAGGCTAGC3`) and MiSeq_SERT_1080_R (5`GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGAAAAGAAGATTTGCGCGG3`).
Primer pair (NT Library Fragment 2): MiSeq_SERT_191_F (5`TCTTTCCCTACACGACGCTCTTCCGATCTGATACACGGCACAGCATTC3`) and MiSeq_SERT_902_R (5`GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGATGTCTTTACTCCCTTCCAG3`).
Primer pair (NT Library Fragment 3): MiSeq_SERT_426_F (5`TCTTTCCCTACACGACGCTCTTCCGATCTGTATCCCGCTCTTCTACATG3`) and MiSeq_SERT_698_R (5`GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAGTCCTCCTCGCTAATCAG3`).
Primer pair (CTLibrary Fragment 1): MiSeq_SERT_1041_F (5`TCTTTCCCTACACGACGCTCTTCCGATCTGGCAGAAGCTTCTTGAAACC3`) and MiSeq_SERT_1592_R (5`GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATCAATGCCACGGTCAGAAC3`).
Primer pair (CT Library Fragment 2): MiSeq_SERT_1250_F (5`TCTTTCCCTACACGACGCTCTTCCGATCTGCTGAGATGAGGAACGAAGA3`) and MiSeq_SERT_1808_R (5`GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGCCCAGGATAATTGACCAG3`).
Primer pair (CT Library Fragment 3): MiSeq_SERT_1376_F (5`TCTTTCCCTACACGACGCTCTTCCGATCTGATGCTGATAACCCTTGG3`) and MiSeq_pCEP4_R (5`GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTATCATGTCTGGATCCGG3`).
In a second round of PCR (15 cycles), Illumina adaptors and experiment-specific barcodes were added.
Primer pair for adding I5 and I7 Illumina adaptors: MiSeq_Start_Adaptamer (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT) and MiSeq_Index_Adaptamer (CAAGCAGAAGACGGCATACGAGAT-6nt-barcode-GTGACTGGAGTTCAGACGTGTGCTCTTC)
Amplicons were deep sequenced with MiSeq v3 and HiSeq 2500 instruments using a 2x250 nt paired end protocol, though data is analyzed as single reads.

treatment_protocol: Cells expressing DNA libraries of SERT mutants (cloned into a modified version of pCEP4, Invitrogen, with an upstream chimeric intron were FACS sorted for expression (the ability of an antibody to bind to an epitope tag in SERT) or transport a flourescent ligand (APP+).  Two single site-saturation mutagenesis libraries were created, spanning residues 2-349(NT Library), 350-630 (CT Library).

tag: protein

value: SERT

tag: sort gate

value: Top 50% of population for expression (1/200anti-myc)

tag: # collected cells


title: 7626

tag: Sex

value: male

tag: sequencing batch

tag: transcript integrity number


title: 077_SCT80_AIT_015_Bcells_E10


title: 10081

tag: age

tag: bmi


title: 6327


title: Expression Sorted  CT Library; Replicate 1


title: 7352


title: KLFb93: HDF_Patient_93


title: 084_SCT80_AIT_015_Bcells_D11


title: 083_SCT80_AIT_015_Bcells_C11

value: yes


title: 075_SCT80_AIT_015_Bcells_C10


title: 076_SCT80_AIT_015_Bcells_D10


title: 074_SCT80_AIT_015_Bcells_B10


title: 7295


title: 072_SCT80_AIT_015_Bcells_H9


title: 073_SCT80_AIT_015_Bcells_A10


title: 071_SCT80_AIT_015_Bcells_G9


title: 7819


title: 7136


title: 7222


title: 7126


title: IonXpress_RNA_55

city: Memphis

first: Quynh

last: Tran

state: TN

address: 262 Danny Thomas Place, C4029, Mail Stop 250

department: Pathology

institute: St. Jude Children's Research Hospital

data_processing:
Aligned first via tophat v2.1.0, allowing for 5 mismatches and setting strandedness to second strand
Took unmapped reads and re-alined with bowtie v2.2.5 under local very sensitive settings to get the remaining ~40% of reads mapped
Merged the tophat and bowtie results files into merged.bam
Counts were generated using Htseq v0.6.0 with parameters stranded=yes, mode=intersection-nonempty with default cutoff score of 10.
Genome_build: GRCm38
Supplementary_files_format_and_content: tab-delimited text files include htseq counts for each Sample.

source_name: Quadriceps

extract_protocol:
RNAs were extracted using PureLink RNA Mini kit by Life Tech
1ug of total RNA was enriched for Poly A RNA using Ambion Dynabeads mRNA Direct Micro kit and barcoded libraries for sequencing were prepared using the Life Technologies RNAseq V2 kit for Ion Torrent according to manufacturer’s standard protocol. Libraries were amplified 14 cycles and the quality of each library was checked on an Agilent Bioanalyzer DNA High Sensitivity chip. A 1ul aliquot of each sample was pooled and run on an Ion Torrent PGM 314 chip. The libraries were equally pooled based on PGM 314 read data, purified on a Pippin Prep gel, quantified by the Agilent Bioanalyzer and sequenced on an Ion Torrent Proton sequencer.

tag: strain

tag: feeding 16h before sacrifice

value: Fasted

tag: diet during week 6-7

value: High Fat Diet

value: Quadriceps

value: 22 weeks of age


title: IonXpress_RNA_061

value: Fed


title: IonXpress_RNA_56


title: IonXpress_RNA_54

value: Control Diet


title: IonXpress_RNA_57


title: IonXpress_RNA_059


title: IonXpress_RNA_064


title: IonXpress_RNA_060


title: Expression Sorted  NT Library; Replicate 1


title: 070_SCT80_AIT_015_Bcells_F9


title: IonXpress_RNA_58


title: IonXpress_RNA_062


title: IonXpress_RNA_53


title: 7096

value: batch 2


title: IonXpress_RNA_063


title: SERT Naïve CT Library

description:
plasmid DNA
Naïve SERT CT SSM library (a.a.350-654)


title: SERT Naïve NT Library

description:
plasmid DNA
Naïve SERT NT SSM library (a.a. 2-349)


title: Transport Sorted  CT Library; Replicate 2

description: Deep mutational scan of SERT for transport

value: Top 50% of population for expression (1/200anti-myc) and top 15% of Transporters


title: 069_SCT80_AIT_015_Bcells_E9


title: IonXpress_RNA_51


title: 7021


title: IonXpress_RNA_52


title: IonXpress_RNA_50


title: 068_SCT80_AIT_015_Bcells_D9


title: 6922


title: IonXpress_RNA_49


title: IonXpress_RNA_48


title: 4510


title: IonXpress_RNA_47


title: 066_SCT80_AIT_015_Bcells_B9


title: 065_SCT80_AIT_015_Bcells_A9


title: 064_SCT80_AIT_015_Bcells_H8


title: IonXpress_RNA_044

source_name: eWAT

value: epididymal white adipose tissue


title: IonXpress_RNA_043


title: 6875


title: IonXpress_RNA_046


title: 6886


title: 6851


title: IonXpress_RNA_040


title: IonXpress_RNA_041


title: 062_SCT80_AIT_015_Bcells_F8

value: blood


title: 2100


title: 063_SCT80_AIT_015_Bcells_G8

value: peripheral blood mononuclear cells


title: IonXpress_RNA_039


title: IonXpress_RNA_042


title: IonXpress_RNA_045


title: IonXpress_RNA_036


title: IonXpress_RNA_038


title: 7366


title: 1726


title: IonXpress_RNA_035


title: 061_SCT80_AIT_015_Bcells_E8

value: B cell


title: IonXpress_RNA_037


title: IonXpress_RNA_034


title: 067_SCT80_AIT_015_Bcells_C9

value: no


title: 060_SCT80_AIT_015_Bcells_D8


title: IonXpress_RNA_032


title: 6632


title: 6639


title: 058_SCT80_AIT_015_Bcells_B8


title: IonXpress_RNA_031


title: IonXpress_RNA_030


title: IonXpress_RNA_033


title: IonXpress_RNA_029


title: 6564


title: 7226


title: 057_SCT80_AIT_015_Bcells_A8


title: 056_SCT80_AIT_015_Bcells_H7


title: IonXpress_RNA_026


title: 6469


title: 562

data_processing:
Reads were aligned to the mouse genome using TopHat v2.0.10 and bowtie v1.0.0 with these parameters  -p 11 --bowtie1 --color --qual
Counts were generated using HTSeq v0.5.4p5 with parameters --strand=no --mode=intersection-nonempty
Genome_build: GRCm38.75
Supplementary_files_format_and_content: text file with read counts.

source_name: Gastrocnemius muscle

extract_protocol:
Gastrocnemius muscles were carefully removed, visibly cleared of any adipose or connective tissue and immediately flash frozen in liquid nitrogen. RNA was extracted using TRIzol reagent and column purified using a PureLink RNA mini kit. Sample quality was determined using an Agilent bioanalyzer. Sequencing libraries were prepared from 1 ug of the purified RNA extract.
500ng of total RNA was enriched for Poly (A) RNA and then barcoded libraries for sequencing were prepared using the Lexogen mRNA Sense kit for Ion Torrent according to manufacturer’s standard protocol. Libraries were amplified 13 cycles and the quality of each library was checked on an Agilent Bioanalyzer DNA High Sensitivity chip. A 1ul aliquot of each sample was pooled and run on the Ion Torrent PGM 314 chip. The libraries were then pooled based on read counts from the PGM 314 chip, purified on a Pippin Prep gel, quantified by the Agilent Bioanalyzer and sequenced on an ABI 5500 Wildfire sequencer.

tag: background strain

tag: genotype

value: Male

value: Gastrocnemius muscle


title: 555

tag: tissue


title: 055_SCT80_AIT_015_Bcells_G7

tag: cell type


title: IonXpress_RNA_028


title: 1698

source_name: subcutaneous adipose tissue

value: male


title: 549

value: fl/fl; Tg/+


title: 054_SCT80_AIT_015_Bcells_F7

tag: cell subtype


title: IonXpress_RNA_027

value: Fasted


title: 540


title: 5949


title: 525


title: 052_SCT80_AIT_015_Bcells_D7

tag: true ige producing memory b cells


title: 5668


title: 516


title: 059_SCT80_AIT_015_Bcells_C8


title: 051_SCT80_AIT_015_Bcells_C7


title: 5481


title: 506


title: 508


title: 5386


title: 479


title: 493


title: 504


title: 049_SCT80_AIT_015_Bcells_A7


title: 048_SCT80_AIT_015_Bcells_H6


title: 050_SCT80_AIT_015_Bcells_B7


title: 5283


title: 437


title: 435


title: 047_SCT80_AIT_015_Bcells_G6


title: 523


title: 5242


title: 5166


title: 398


title: 045_SCT80_AIT_015_Bcells_E6


title: 046_SCT80_AIT_015_Bcells_F6


title: 397


title: 399_2


title: 5135


title: 044_SCT80_AIT_015_Bcells_D6


title: 491


title: 369


title: 5365


title: 432


title: 373


title: 366


title: 399_1


title: 364


title: 402


title: 404


title: 042_SCT80_AIT_015_Bcells_B6


title: 344


title: 2


title: 041_SCT80_AIT_015_Bcells_A6


title: 039_SCT80_AIT_015_Bcells_G5


title: 4821


title: 2877


title: 036_SCT80_AIT_015_Bcells_D5


title: 4479


title: 4986


title: 037_SCT80_AIT_015_Bcells_E5


title: 4420


title: 5983


title: 4455


title: 4219


title: 034_SCT80_AIT_015_Bcells_B5


title: 043_SCT80_AIT_015_Bcells_C6


title: 033_SCT80_AIT_015_Bcells_A5


title: 11267


title: 4856


title: 031_SCT80_AIT_015_Bcells_G4


title: 030_SCT80_AIT_015_Bcells_F4


title: 4148


title: 4000


title: 029_SCT80_AIT_015_Bcells_E4


title: 038_SCT80_AIT_015_Bcells_F5


title: 3944


title: 4154


title: 032_SCT80_AIT_015_Bcells_H4


title: 7111


title: 3985


title: 027_SCT80_AIT_015_Bcells_C4


title: 024_SCT80_AIT_015_Bcells_H3


title: 023_SCT80_AIT_015_Bcells_G3


title: 2912


title: 026_SCT80_AIT_015_Bcells_B4


title: 025_SCT80_AIT_015_Bcells_A4


title: 2886


title: 028_SCT80_AIT_015_Bcells_D4


title: 2391


title: 035_SCT80_AIT_015_Bcells_C5


title: 019_SCT80_AIT_015_Bcells_C3


title: 2871


title: 022_SCT80_AIT_015_Bcells_F3


title: 018_SCT80_AIT_015_Bcells_B3


title: 2892


title: 021_SCT80_AIT_015_Bcells_E3


title: 3995


title: 2748


title: 2741


title: 4589


title: 017_SCT80_AIT_015_Bcells_A3

value: yes


title: 2769


title: 016_SCT80_AIT_015_Bcells_H2


title: 7305


title: 012_SCT80_AIT_015_Bcells_D2


title: 2714


title: 2736


title: 014_SCT80_AIT_015_Bcells_F2


title: 013_SCT80_AIT_015_Bcells_E2


title: 2605


title: 011_SCT80_AIT_015_Bcells_C2


title: 2590


title: 2599


title: 010_SCT80_AIT_015_Bcells_B2


title: 2583


title: 015_SCT80_AIT_015_Bcells_G2


title: 2902


title: 009_SCT80_AIT_015_Bcells_A2


title: 2580


title: 008_SCT80_AIT_015_Bcells_H1


title: 007_SCT80_AIT_015_Bcells_G1


title: 2514


title: 2513


title: 006_SCT80_AIT_015_Bcells_F1


title: 10530


title: 005_SCT80_AIT_015_Bcells_E1


title: 10062


title: Lung_fibroblast_IPF_TGFb_24h_rep2 [Illumina]

type: genomic

city: Ciudad de Mexico

first: Yalbi

middle: Itzel

last: Balderas-Martinez

state: Distrito Federal

address: Calzada de Tlalpan 4502, Sección XVI, Belisario Domínguez

country: Mexico

institute: INER

description: Gene expression data from IPF fibroblast

hyb_protocol: Bisulfite converted DNA was amplified, fragmented, and hybridized to Illumina Infinium Humanmethylation450 BeadChip (Catalog: WG-314-1003) using standard Illumina protocol. Amplification of bisulfite converted DNA and hybridization was done in the Illumina Hybridization Oven.

scan_protocol: After hybridization, BeadChips were washed, and primers hybridized to the samples were extended, and stained following Illumina standard protocol. BeadChips were imaged in the Illumina HiScan system, using the Methylation NXT scan setting.

data_processing: Data were analyzed using R 3.4.2 (http://www.r-project.org/), using Bioconductor 3.6 (http://www.bioconductor.org/), following the pipeline of Maksimovic (PMID:27347385). For all probed genomic positions, detection p-values >0.01 were filtered out (485,179 loci). Probes with SNPs at CpG or SBE site were filtered (456,352), followed by filtering of probes that have shown to be cross-reactive, leaving 429,792 probes used for analysis.

label: Cy5 and Cy3

source_name: IPF, TGFb24h

label_protocol: 500 ng of DNA sample was used for bisulfite convertion using the standard Illumina Infinium HD Methylation method (Illumina Document # 15019519 v04

growth_protocol: All primary cultures were grown in Ham’s F-12 medium with 10% FBS at 37 °C in an atmosphere of 95% air and 5% CO2 until early confluence

extract_protocol: Genomic DNA extraction was performed with MasterPure Complete DNA and RNA purification Kit (Epicentre) following the manufacturer’s instructions.

treatment_protocol: Cells were treated with TGF-β1 (10 ng/ml), using two different time periods (24 hours or 5 days) all of them cultured in HAM´s F-12 with low serum (0.1%).

value: Lung

tag: disease state

value: IPF

tag: treatment


title: 2469

extract_protocol:
Subcutaneous adipose needle biopsies were obtained from near the umbilicus of men from the METSIM cohort. RNA was isolated using the QIAGEN miRNeasy kit.
Libraries were constructed using a polyA+ selection protocol (Illumina TruSeq RNA Sample Preparation Kit v2)


title: Lung_fibroblast_IPF_TGFb_5d_rep1 [Illumina]

source_name: IPF, TGFb5d


title: 002_SCT80_AIT_015_Bcells_B1


title: 2436


title: 8100


title: 001_SCT80_AIT_015_Bcells_Control_A1


title: VEH

city: Columbia

first: Wurood

last: Neamah

state: SC

address: 6439 Garners Ferry Ro., Bld #1/C7

institute: School Of Medicine

hyb_protocol: Labeled samples were hybridized to miRNA 4.0 chips overnight (16h, 48°C, 60 RPM) then washed and stained on an Affymetrix GCS 3000 system Fluidics station using the FS450_0002 protocol.

scan_protocol: Chips were scanned on a Affymetrix GCS 3000 system scanner.

data_processing:
Affymetrix Expression Console Version 1.4.1.46 was used for quality control, data summarization, and normalization. The processing algorithm used was RMA-DABG. Affymetrix Transcriptome Analysis Console Version 3.1.0.5 was used to perform differential expression analyses. MicroRNAs were considered differentially expressed between the two groups if linear fold change was greater than ±2 and the ANOVA p-value was less than 0.05
affymetrix-algorithm-name = rma : dabg
affymetrix-algorithm-version = 1.0
affymetrix-array-type = miRNA-4_0
program-name = Expression Console
program-version = 1.4.1.46

label: Biotin

source_name: Peritoneal cavity_vehicle

label_protocol: 500ng total RNA was used as starting material for labeling according to the Affymetrix FlashTag Biotin HSR RNA Labeling Kit manufacturer protocol.

growth_protocol: Mice were injected  with 10 µg/kg TCDD or VEH

extract_protocol: Total RNA was isolated with Qiagen miRNeasy micro kit.

treatment_protocol: Cells were collected after 3 days of TCDD treatment

tag: gender

value: Female

value: 3 days

value: vehicle

value: Peritoneal cavity


title: TCDD

source_name: Peritoneal cavity_TCDD

value: 10 µg/kg TCDD


title: Lung_fibroblast_normal_TGFb_5d_rep2 [Illumina]

description: Gene expression data from normal fibroblast

source_name: normal, TGFb5d

value: normal


title: Uninfected_3_240

city: Jena

first: Philipp

last: Kämmer

address: Adolf-Reichwein-Straße 23

department: Microbial Pathogenicity Mechanisms

country: Germany

institute: Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute (HKI)

data_processing:
RTA software version 1.18.64 were used for basecalling.
Read quality were checked with Fastqc. Reads were trimmed with trimmomatic version 0.32 and following parameters: SE threads 5 -phred33 FILE LEADING: 3 TRAILING: 3 SLIDINGWINDOW: 15:20 MINLEN 30
Trimmed reads were mapped to reference genome of Candida albicans A22-s05-m04-r04_features and of Homo sapiens GRCh38.77 using tophat2 with following parameters: -p 10 -g 1 --no-mixed --no-discordant --b2-very-sensitive --max-intron-length 100000 -o OUTDIR -G ANNOTATION INDEX FASTQFILE > OUTFILE
Mapped reads were counted using featureCounts version 1.4.5 and DeSeq2 were used for differential expression filtering.
Genome_build: GRCh38.77, A22_2015-Apr-28
Supplementary_files_format_and_content: Comma-delimited text files include counts for each gene per sample

source_name: whole blood

growth_protocol: Because 1× PBS was used for mock infection samples, there is no growth protocol for mock infected samples.

extract_protocol:
To isolate human RNA aliquots of infected or mock infected samples were added to a PAXgene® Blood RNA Tube (PreAnalytiX) and processed with the PAXgene® Blood RNA Kit (PreAnalytiX) according to the manufacturer’s protocol.  The cell pellet was further processed with the RiboPure™-Yeast Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. RNA quantity was determined with NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific) and RNA quality was verified with an Agilent 2100 Bioanalyzer (Agilent Technologies). Human RNA samples were pooled subsequently in a quantitative ratio of 1:10.
Library Preperation was performed by GATC Biotech using validated  standard procedures modified to optimize the method for automated  Preperation. Library Preparation consisted of the following steps:  Purification of poly-A containing mRNA molecules, mRNA fragmentation, Random primed cDNA synthesis (strand specific), Adapter ligation and adapter specific PCR amplification

tag: time point

value: 240 minutes


title: Lung_fibroblast_IPF_TGFb_5d_rep2 [Illumina]


title: 2421

data_processing:
Fastx toolkit was used to remove low quality reads; reads with phred quality > 20 for 80% of bases were retained.
Tagdust was used to identify and remove reads containing adapter and linker sequences.
STAR (version 2.4.2a) was used as the aligner, with hg19 and GENCODE v19 as annotation; STAR was run, using a 2-pass strategy, were splice locations from the first pass were used as additional annotation for the second pass.
salmon (version 0.7.2) was used to estimate isoform abundance from aligned BAM files; GENCODE 19 was used as annotation. --gcBias option was used.
tximport (version 1.0.3) was used to collapse isoform abundance estimates from salmon to gene abuddance estimates
Genome_build: hg19
Supplementary_files_format_and_content: Abundance measurements generated using salmon and tximport; values represent the transcripts per million (TPM) estimate of gene abundance.

tag: Sex

tag: sequencing batch

value: batch 2


title: control3

city: Wuhan

first: Libin

last: Yan

state: Hubei

address: No.1095, Jiefang Avenue

department: Urology

institute: Tongji Hospital,Tongji Medical College, Huazhong University of Science Technology

description: si-NC

hyb_protocol: standard Agilent protocol

scan_protocol: standard Agilent protocol

data_processing: standard Agilent protocol

source_name: A498 cell line

label_protocol: standard Agilent protocol

extract_protocol: none provided by the submitter

tag: cell line

value: control


title: Lung_fibroblast_IPF_control_rep1 [Illumina]

source_name: IPF, control


title: Aza

description:
Aza treated hTERT replicate one
SAMPLE 4

hyb_protocol: Bisulphite converted DNA was amplified, fragmented and hybridised to Illumina Infinium Human Methylation450k Beadchip using standard Illumina protocol.

scan_protocol: Arrays were imaged using BeadArray Reader using standard recommended Illumina scanner setting.

data_processing: BeadStudio software v3.2 and tailored RnBeads scripts through Rstudio

source_name: hTERT

label_protocol: Standard Illumina Protocol

growth_protocol: The parental or wild type (WT) adherent  hTERT1604 lung fibroblast cell line was cultured in 4.5g/l glucose DMEM supplemented with 10% FBS and 2x NEAA. Aza and DNMT1 targeting siRNA treated hTERT1604 cells were maintained as per WT.

extract_protocol: The genomic DNA isolated was assessed for purity and integrity prior to quantification using the Picogreen fluorescent assay.

treatment_protocol: Aza treatment was performed by seeding hTERT-1604 cells onto a 90 mm plate in medium (as per WT) and allowed to attach overnight before replacing with complete medium containing 1 μM Aza, which was renewed at 24-hour intervals up to 72 hours. siRNA treatments were carried out in 6-well plates, each well seeded with 1×106 hTERT-1604 cells before reverse transfection using Dharmafect and 100 nM (hTERT-1604) ON-TARGETplus SMARTpool DNMT1 siRNA or a matched concentration of scrambled control. Cells were cultured in complete medium until time appropriate pellets could be collected.

value: Male


title: Lung_fibroblast_normal_TGFb_24h_rep2 [Illumina]

source_name: normal, TGFb24h


title: DNMT1_siRNA_2

description:
DNMT1 targeting siRNA treated WThTERTS replicate two
SAMPLE 7


title: Lung_fibroblast_normal_control_rep2 [Illumina]

source_name: normal, control


title: 2375

tag: transcript integrity number

tag: age

tag: bmi


title: 11282


title: case2


title: Aza_1

description:
Aza treated hTERT replicate two
SAMPLE 5


title: Uninfected_1_240


title: Uninfected_2_240


title: 040_SCT80_AIT_015_Bcells_H5

source_name: B cell single cell seq

value: blood

value: peripheral blood mononuclear cells

value: B cell

value: no


title: 2403

value: male


title: 003_SCT80_AIT_015_Bcells_C1


title: WT_3

description:
WThTERT replicate three
SAMPLE 3


title: DNMT1_siRNA

description:
DNMT1 targeting siRNA treated WThTERTS replicate one
SAMPLE 6


title: WT_2

description:
WThTERT replicate two
SAMPLE 2


title: WT_1

description:
WThTERT replicate one
SAMPLE 1


title: control1

description: si-NC


title: Lung_fibroblast_normal_control_rep1 [Illumina]

value: Lung


title: Expression Sorted  NT Library; Replicate 2

source_name: Synthetic coding sequences

tag: protein

value: SERT

tag: sort gate

value: Top 50% of population for expression (1/200anti-myc)

tag: # collected cells


title: case1


title: control2

description: si-NC


title: Lung_fibroblast_IPF_control_rep2 [Illumina]

tag: tissue

value: IPF


title: 2264


title: Lung_fibroblast_normal_TGFb_5d_rep1 [Illumina]


title: 2262


title: 004_SCT80_AIT_015_Bcells_D1

tag: cell type


title: Transport Sorted  NT Library; Replicate 2

value: Top 50% of population for expression (1/200anti-myc) and top 15% of Transporters


title: 2260


title: STL001.TH1.ADS124

city: Daejeon

first: Inkyung

last: Jung

address: KAIST, 291 Daehak-ro, Yuseong-gu

department: Biological Sciences

country: South Korea

institute: KAIST

description:
TH.pp.all.bz2
TH.po.all.bz2

data_processing:
Read pairs from Promoter Capture Hi-C library were independently mapped to human genome hg19 using BWA-mem and manually paired with in house script.
Unmapped, non-uniquely mapped, and PCR duplicates were removed.
Trans-chromosomal read pairs and putative self-ligated products (<15kb read pairs) were removed.
Off-target reads were removed when both read pairs did not match to capture probe sequences.
Genome_build: Hg19
Supplementary_files_format_and_content: *.po and *.pp provide interaction frequencies for promoter-promoter interactions or promoter-other interactions, respectively.  We combined multiple donors or replicates according to cell/tissue types.

source_name: Human Thymus

extract_protocol:
Promoter Capture Hi-C library was constructed by performing a target enrichment protocol (enriching target promoter-centered proximity ligation fragments from Hi-C library using capture RNA probes). Briefly, we incubated 500ng Hi-C library 24h at 65 °C within a humidified hybridization chamber with 2.5ug human Cot-1 DNA (Life Technologies), 2.5ug salmon sperm DNA (Life Technologies), and p5/p7 blocking oligos with hybridization buffer mix (10X SSPE, 10mM EDTA, 10X Denhardts solution, and 0.26% SDS) and 500ng RNA probes. RNA:DNA hybrids were enriched using 50ul T1 streptavidin beads (Invitrogen) with 30min incubation at RT. Next, bead-bound hybrids were washed by a 15min incubation in wash buffer1 (1X SSC and 0.1% SDS) with frequent vortexing, and then washed three times with 500ul of pre-warmed (65 °C) wash buffer2 (0.1X SSC and 0.1% SDS), and resuspended in nuclease-free water. The resulting capture Hi-C libraries were amplified while bound to the T1 beads, and purified using AMPure XP beads, followed by sequencing.
Promoter Capture Hi-C library sequencing procedures were carried out as previously described according to Illumina HiSeq2500 or HiSeq4000 protocols with minor modifications (Illumina, San Diego, CA).
Capture Hi-C

value: Thymus


title: STL003.SX3.ADS390

description:
SX.pp.all.bz2
SX.po.all.bz2

source_name: Human Spleen

value: Spleen


title: 2226

first: Karen

last: Mohlke

address: 120 Mason Farm Rd

institute: The University of North Carolina at Chapel Hill


title: STL001.SX1.ADS374

description:
SX.pp.all.bz2
SX.po.all.bz2


title: ∆ctx rep1 (cecum)

city: Boston

first: Fabian

last: Rivera

state: UNITED STATES

address: 77 Avenue Louis Pasteur, NRB 1039

department: Microbiology

institute: Harvard Medical School

description:
Infection with Vibrio cholerae
Cecum.xlsx

data_processing:
Sequencing of the cDNA libraries was performed by the Biopolymers Facility at Harvard Medical School. RNA-sequencing library quantification was performed on HiSeq 2500 Rapid PE V2 by 100 bp paired-end high-throughput sequencing.
The raw reads in FASTQ format were aligned to chromosomes I and II of the V. cholerae El Tor N16961 genome (RefSeq accession numbers NC_002505 and NC_002506) using the bowtie2 algorithm on Geneious 10.0 (http://www.geneious.com).
Differential expression analysis was performed on Geneious using DEseq 2
Genome_build: NC_002505 and NC_002506
Supplementary_files_format_and_content: WT ileum vs cecum.xlsx: Excel file containing log2 differential expression of wild type V. cholerae C6706 ORFs in the Ileum relative to the cecum.
Supplementary_files_format_and_content: Ileum.xlsx: Excel file containing log2 differential expression of ORFs in the wild type relative to the ∆ctx mutant during infection of the ileum; RPKMs.
Supplementary_files_format_and_content: Cecum.xlsx: Excel file containing log2 differential expression of ORFs in the wild type relative to the ∆ctx mutant during infection of the cecum; RPKMs.

organism: Vibrio cholerae

source_name: Cecum of 3-day old infant rabbits infected with V. cholerae

extract_protocol:
Luminal samples from the ileum and cecum of 3-day old infant rabbits infected with V. cholerae were snap-frozen in liquid nitrogen for subsequent phenol:chloroform RNA isolation. Contaminating DNA was removed using the DNA-free kit (Applied Biosystems) and RNA was stored at -80°C.
The Ovation Complete Prokaryotic RNA-Seq Library Systems (NuGEN) was used for cDNA synthesis and library preparation of 200 bp (mean size) fragments. cDNA library quantification was performed using the Agilent 2200 TapeStation.

tag: breed (host)

value: New Zealand rabbit

tag: tissue (host)

value: Cecum

tag: genotype

value: {delta}ctx bacteria

tag: time

value: one-day post-intragastric infection


title: ∆ctx rep2 (ileum)

description:
Infection with Vibrio cholerae
Ileum.xlsx

source_name: Ileum of 3-day old infant rabbits infected with V. cholerae

value: Ileum


title: WT rep1 (cecum)

description:
Infection with Vibrio cholerae
WT ileum vs cecum.xlsx
Cecum.xlsx

value: wild type bacteria


title: 2198

city: Chapel Hill

source_name: subcutaneous adipose tissue

value: batch 2


title: 627


title: ∆ctx rep1 (ileum)


title: WT rep2 (ileum)

description:
Infection with Vibrio cholerae
WT ileum vs cecum.xlsx
Ileum.xlsx


title: 5532


title: 2131


title: STL002.SB2.ADS128

description:
SB.pp.all.bz2
SB.po.all.bz2

source_name: Human Small Bowel

value: Small Bowel


title: Transport Sorted  NT Library; Replicate 1


title: WT rep1 (ileum)


title: WT rep2 (cecum)


title: Lung_fibroblast_normal_TGFb_24h_rep1 [Illumina]

tag: disease state

value: normal

tag: treatment


title: STL001.SG1.ADS228

description:
SG.pp.all.bz2
SG.po.all.bz2

source_name: Human Sigmoid Colon

value: Sigmoid Colon


title: STL003.RV3.ADS462

description:
RV.pp.all.bz2
RV.po.all.bz2

source_name: Human Right Ventricle

value: Right Ventricle


title: 2129

tag: Sex

value: male


title: 2113

tag: sequencing batch


title: STL003.RV3.ADS132

description:
RV.pp.all.bz2
RV.po.all.bz2


title: abK4me  ChIP-Seq SCP3 positive H1t negative nuclei

first: Kevin

last: Brick

state: MD

address: 5/205 Memorial Drive

department: GBB

institute: NIDDK

data_processing:
BigWig files were generated in one of four ways:
1. BIGWIG files named: monoCorrected.ws25bp.bigwig:
Coverage normalized by calibration with internal spike-in. We retained only sequencing reads with an alignment q-score >= 20, that were mapped in a “proper pair” (for paired-end data), that passed vendor QC and that were mapped as a primary alignment. For paired-end data, we only retained reads from fragments with an insert size between 100 bp and 200 bp. We first converted paired-end reads to fragments, then calculated the enrichment of fragments mapping to each spike-in sequence after ChIP. Normalization using the K-MetStat spike-in was performed as described previously (Grzybowski et al. 2015), with slight modification (see methods of the paper linked to this submission; Lam et al. 2019).
2. BIGWIG files named: ncisCorrectedByStablePromoters.ws25bp.bigwig
We retained only sequencing reads with an alignment q-score >= 20, that were mapped in a “proper pair” (for paired-end data), that passed vendor QC and that were mapped as a primary alignment. We also only retained reads from fragments with an insert size between 100 bp and 200 bp. We first converted paired-end reads to fragments. Read coverage in non-overlapping 25bp windows of the genome was calculated. NCIS and input DNA of a matched experiment were used to estimate a background normalization factor for each experiment. The NCIS-normalized input DNA coverage was subtracted from the count in each 25bp window. We next identified a subset of stable gene promoters that varied little across the five populations of meiotic prophase (see methods of the paper linked to this submission; Lam et al. 2019). The coverage in each 25bp window was divided by the mean coverage at stable promoters.
3. BIGWIG files named: 150bp.RPKM.bigwig
We used deeptools 3.0.1 and the following arguments: bamCoverage -b $bam -o $bigwig --binSize 150 --ignoreForNormalization chrX chrY chrM --normalizeUsing RPKM
4. BIGWIG files named: NCIS.ws25bp.bigwig
Read coverage in non-overlapping 25bp windows of the genome was calculated. NCIS and input DNA were used to estimate a background normalization factor for each experiment. The NCIS-normalized input DNA coverage was subtracted from the count in each 25bp window.
Genome_build: mm10
Supplementary_files_format_and_content: Peaks for H3K4me3 ChIP-Seq were called using MACS2 (version 2.1.2) with default parameters except (-q 0.1 --broad) and with a stage-matched input DNA sample as a control.
Supplementary_files_format_and_content: BigWig files were generated in one of four ways:   1. BIGWIG files named: monoCorrected.ws25bp.bigwig:   Coverage normalized by calibration with internal spike-in. We retained only sequencing reads with an alignment q-score >= 20, that were mapped in a “proper pair” (for paired-end data), that passed vendor QC and that were mapped as a primary alignment. For paired-end data, we only retained reads from fragments with an insert size between 100 bp and 200 bp. We first converted paired-end reads to fragments, then calculated the enrichment of fragments mapping to each spike-in sequence after ChIP. Normalization using the K-MetStat spike-in was performed as described previously (Grzybowski et al. 2015), with slight modification (see methods of the paper linked to this submission; Lam et al. 2019).    2. BIGWIG files named: ncisCorrectedByStablePromoters.ws25bp.bigwig  We retained only sequencing reads with an alignment q-score >= 20, that were mapped in a “proper pair” (for paired-end data), that passed vendor QC and that were mapped as a primary alignment. We also only retained reads from fragments with an insert size between 100 bp and 200 bp. We first converted paired-end reads to fragments. Read coverage in non-overlapping 25bp windows of the genome was calculated. NCIS and input DNA of a matched experiment were used to estimate a background normalization factor for each experiment. The NCIS-normalized input DNA coverage was subtracted from the count in each 25bp window. We next identified a subset of stable gene promoters that varied little across the five populations of meiotic prophase (see methods of the paper linked to this submission; Lam et al. 2019). The coverage in each 25bp window was divided by the mean coverage at stable promoters.    3. BIGWIG files named: 150bp.RPKM.bigwig  We used deeptools 3.0.1 and the following arguments: bamCoverage -b $bam -o $bigwig --binSize 150 --ignoreForNormalization chrX chrY chrM --normalizeUsing RPKM     4. BIGWIG files named: NCIS.ws25bp.bigwig  Read coverage in non-overlapping 25bp windows of the genome was calculated. NCIS and input DNA were used to estimate a background normalization factor for each experiment. The NCIS-normalized input DNA coverage was subtracted from the count in each 25bp window.

extract_protocol:
Chromatin extracted for ChIP-Seq was either sheared by sonication or Micrococcal nuclease (MNase) digestion. Chromatin for all H3K4me3 ChIP-Seq for studying histone dynamics in stage-specific populations was sheared by sonication, whereas ChIP-Seq for identifying histone marks at hotspots were performed on MNase-digested chromatin. Experimental procedures are listed below in detail.
Shearing chromatin by sonication:
Frozen nuclei pellets were thawed at RT for 10 min. Nuclei were lysed with Lysis buffer 1 (0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl pH 8) and incubated at RT for 10 min. Nuclei pellets were subsequently washed with Lysis buffer 2 (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl pH 8) and lysed with RIPA buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl plus protease inhibitor). Chromatin was sheared into ~500−1000 bp fragments by sonication using Bioruptor (diagenode). Chromatin concentration was measured using a Qubit dsDNA HS Assay Kit (Thermo Fisher).
MNase digestion:
Frozen nuclei pellets were thawed at RT for 10 min. Nuclei pellets were resuspended with MNase buffer (50 mM Tris-HCl pH 8, 1 mM CaCl2, 4 mM MgCl2, 4% NP-40 plus protease inhibitor). MNase digestion was performed in a concentration of 3U MNase (USB, Affymetrix) per one million nuclei at 37°C for 5 min. The reaction was stopped by adding a final concentration of 10 mM EDTA and incubated at 4°C for 5 min. Chromatin was pelleted and resuspended in RIPA buffer. Chromatin concentration was measured.
For stage-specific H3K4me3-ChIP-Seq, modified mononucleosomes (SNAP-ChIP K-MetStat Panel, EpiCypher) were added at a spike-in volume of 1 μl per ~1.5 μg chromatin DNA. Chromatin was immunoprecipitated with 0.8-5 μg antibodies (or 5 μl unpurified serum) in 0.5-1 ml RIPA buffer at 4°C overnight (see Supplementary Table 1 for details). The immuno-complexes were captured using 20-75 μl Dynabeads Protein G (30 mg/ml, Novex) at 4°C for 2 hr. The beads were washed with buffers previously described27. ChIPed DNA was eluted using a IPure kit v2 (diagenode).
ChIP-seq libraries were constructed with a KAPA Hyper Prep Kit (Kapa Biosystems) following steps for generating 1 μg of library DNA. DNA libraries were cleaned up with an Agencourt AMPure XP system (Beckman Coulter). The DNA concentration and fragment size of these libraries were measured with a Qubit dsDNA HS Assay Kit (Thermo Fisher) and an Agilent High Sensitivity DNA Kit (Agilent), respectively.

treatment_protocol:
Testes from adult mice were de-capsulated and fixed with 1% formaldehyde for 10 min followed by 5 min of quenching at RT. Fixed tissues were homogenized and filtered through a 70- or 100-μm cell strainer. After washing with chilled 1 x PBS, nuclei were extracted using nucleus extraction buffer (15 mM Tris-HCl pH 7.4, 0.34 M sucrose, 15 mM NaCl, 60 mM KCl, 0.2 mM EDTA, 0.2 mM EGTA) on ice for 5 min and were homogenized with 20 strokes with loose pestle followed by 10 strokes with tight pestle. Nuclei were filtered through a 40-μm cell strainer and resuspended in chilled PBTB buffer (1 x PBS with 0.1% Triton X-100, 5% BSA and protease inhibitor).
Nuclei were incubated in 10% of normal serum at RT for 10 min. This blocking step helps reduce non-specific binding of antibodies. Nuclei were then labelled with different combinations of primary antibodies (1 μg of antibodies to 11 million events) in 10% of normal serum at 20°C for 40 min. Nuclei were washed, resuspended in PBTB, blocked with serum, and then labelled with secondary antibodies (1:250 dilution) in 10% of normal serum at 20°C for 30 min. Controls for each secondary antibody were also prepared for setting up the threshold of background fluorescent signal for FACS. Nuclei were washed, resuspended in PBTB, and stored at 4°C until sorting.
Nuclei were filtered through a 40-μm cell strainer, and stained with DAPI for 30 min or longer before sorting. All sorting experiments were performed on either a BD FACSAria II or a BD FACSAria Fusion flow cytometer at a flow rate of ~20,000 events/sec. Singlets were gated using both forward scatter and side scatter. Nuclei from primary spermatocytes (4C) were gated based on DNA content deduced from the DAPI signal, and sorted into populations of interest based on fluorochrome intensity into collection tubes containing PBTB. Sorted nuclei were collected by centrifugation. Nuclei were examined under microscope and counted with a hemocytometer. Finally, excess buffer was removed and nuclei pellets were stored at -80°C.

tag: strain

tag: genotype/variation

value: wild-type

value: adult

value: whole testis

tag: meiotic nuclei population

tag: chip antibody

value: Anti-H3K4me2/3 Abcam (ab8580)


title: 4357

tag: transcript integrity number

tag: age

tag: bmi


title: 7700


title: Input  ChIP-Seq SCP3 positive H1t negative nuclei

description: Input

tag: tissue


title: Transport Sorted  CT Library; Replicate 3

tag: protein

value: SERT

tag: sort gate

value: Top 50% of population for expression (1/200anti-myc) and top 15% of Transporters

tag: # collected cells


title: STL002.SB2.ADS383

description:
SB.pp.all.bz2
SB.po.all.bz2


title: STL003.RA3.ADS375

description:
RA.pp.all.bz2
RA.po.all.bz2

source_name: Human Right Atrium

value: Right Atrium


title: IgG  ChIP-Seq SCP3 positive H1t negative nuclei

description: IgG

value: Anti-IgG Millipore (12-370)


title: H4ac5  ChIP-Seq SCP3 positive H1t negative nuclei

value: Anti-H4ac5 Millipore (06-946)


title: Lung_fibroblast_IPF_TGFb_24h_rep1 [Illumina]

source_name: IPF, TGFb24h

value: Lung

value: IPF


title: ChIP-Seq with no antibody in SCP3 positive STRA8 positive nuclei

description: ChIP-Seq

data_processing:
Sequencing tags were aligned to the mouse mm10 reference genome using BWA mem 0.7.12. For ChIP-Seq samples containing the K-MetStat spike-in panel, we aligned to a custom mm10 genome to which we added the sequences of the DNA bound by each K-MetStat nucleosome.
Genome_build: mm10
Supplementary_files_format_and_content: Peaks for H3K4me3 ChIP-Seq were called using MACS2 (version 2.1.2) with default parameters except (-q 0.1 --broad) and with a stage-matched input DNA sample as a control.
Supplementary_files_format_and_content: BigWig files were generated in one of four ways:   1. BIGWIG files named: monoCorrected.ws25bp.bigwig:   Coverage normalized by calibration with internal spike-in. We retained only sequencing reads with an alignment q-score >= 20, that were mapped in a “proper pair” (for paired-end data), that passed vendor QC and that were mapped as a primary alignment. For paired-end data, we only retained reads from fragments with an insert size between 100 bp and 200 bp. We first converted paired-end reads to fragments, then calculated the enrichment of fragments mapping to each spike-in sequence after ChIP. Normalization using the K-MetStat spike-in was performed as described previously (Grzybowski et al. 2015), with slight modification (see methods of the paper linked to this submission; Lam et al. 2019).    2. BIGWIG files named: ncisCorrectedByStablePromoters.ws25bp.bigwig  We retained only sequencing reads with an alignment q-score >= 20, that were mapped in a “proper pair” (for paired-end data), that passed vendor QC and that were mapped as a primary alignment. We also only retained reads from fragments with an insert size between 100 bp and 200 bp. We first converted paired-end reads to fragments. Read coverage in non-overlapping 25bp windows of the genome was calculated. NCIS and input DNA of a matched experiment were used to estimate a background normalization factor for each experiment. The NCIS-normalized input DNA coverage was subtracted from the count in each 25bp window. We next identified a subset of stable gene promoters that varied little across the five populations of meiotic prophase (see methods of the paper linked to this submission; Lam et al. 2019). The coverage in each 25bp window was divided by the mean coverage at stable promoters.    3. BIGWIG files named: 150bp.RPKM.bigwig  We used deeptools 3.0.1 and the following arguments: bamCoverage -b $bam -o $bigwig --binSize 150 --ignoreForNormalization chrX chrY chrM --normalizeUsing RPKM     4. BIGWIG files named: NCIS.ws25bp.bigwig  Read coverage in non-overlapping 25bp windows of the genome was calculated. NCIS and input DNA were used to estimate a background normalization factor for each experiment. The NCIS-normalized input DNA coverage was subtracted from the count in each 25bp window.

source_name: SCP3 positive STRA8 positive nuclei

extract_protocol:
Chromatin extracted for ChIP-Seq was either sheared by sonication or Micrococcal nuclease (MNase) digestion. Chromatin for all H3K4me3 ChIP-Seq for studying histone dynamics in stage-specific populations was sheared by sonication, whereas ChIP-Seq for identifying histone marks at hotspots were performed on MNase-digested chromatin. Experimental procedures are listed below in detail.     Shearing chromatin by sonication:  Frozen nuclei pellets were thawed at RT for 10 min. Nuclei were lysed with Lysis buffer 1 (0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl pH 8) and incubated at RT for 10 min. Nuclei pellets were subsequently washed with Lysis buffer 2 (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl pH 8) and lysed with RIPA buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl plus protease inhibitor). Chromatin was sheared into ~500−1000 bp fragments by sonication using Bioruptor (diagenode). Chromatin concentration was measured using a Qubit dsDNA HS Assay Kit (Thermo Fisher).    MNase digestion:  Frozen nuclei pellets were thawed at RT for 10 min. Nuclei pellets were resuspended with MNase buffer (50 mM Tris-HCl pH 8, 1 mM CaCl2, 4 mM MgCl2, 4% NP-40 plus protease inhibitor). MNase digestion was performed in a concentration of 3U MNase (USB, Affymetrix) per one million nuclei at 37°C for 5 min. The reaction was stopped by adding a final concentration of 10 mM EDTA and incubated at 4°C for 5 min. Chromatin was pelleted and resuspended in RIPA buffer. Chromatin concentration was measured.
For stage-specific H3K4me3-ChIP-Seq, modified mononucleosomes (SNAP-ChIP K-MetStat Panel, EpiCypher) were added at a spike-in volume of 1 μl per ~1.5 μg chromatin DNA. Chromatin was immunoprecipitated with 0.8–5 μg antibodies (or 5 μl unpurified serum) in 0.5–1 ml RIPA buffer at 4°C overnight (see Supplementary Table 1 for details). The immuno-complexes were captured using 20–75 μl Dynabeads Protein G (30 mg/ml, Novex) at 4°C for 2 hr. The beads were washed with buffers previously described27. ChIPed DNA was eluted using a IPure kit v2 (diagenode).
ChIP-seq libraries were constructed with a KAPA Hyper Prep Kit (Kapa Biosystems) following steps for generating 1 μg of library DNA. DNA libraries were cleaned up with an Agencourt AMPure XP system (Beckman Coulter). The DNA concentration and fragment size of these libraries were measured with a Qubit dsDNA HS Assay Kit (Thermo Fisher) and an Agilent High Sensitivity DNA Kit (Agilent), respectively.

treatment_protocol:
Testes from adult mice were de-capsulated and fixed with 1% formaldehyde for 10 min followed by 5 min of quenching at RT. Fixed tissues were homogenized and filtered through a 70- or 100-μm cell strainer. After washing with chilled 1 x PBS, nuclei were extracted using nucleus extraction buffer (15 mM Tris-HCl pH 7.4, 0.34 M sucrose, 15 mM NaCl, 60 mM KCl, 0.2 mM EDTA, 0.2 mM EGTA) on ice for 5 min and were homogenized with 20 strokes with loose pestle followed by 10 strokes with tight pestle. Nuclei were filtered through a 40-μm cell strainer and resuspended in chilled PBTB buffer (1 x PBS with 0.1% Triton X-100, 5% BSA and protease inhibitor).
Nuclei were incubated in 10% of normal serum at RT for 10 min. This blocking step helps reduce non-specific binding of antibodies. Nuclei were then labelled with different combinations of primary antibodies (1 μg of antibodies to 11 million events) in 10% of normal serum at 20°C for 40 min. Nuclei were washed, resuspended in PBTB, blocked with serum, and then labelled with secondary antibodies (1:250 dilution) in 10% of normal serum at 20°C for 30 min. Controls for each secondary antibody were also prepared for setting up the threshold of background fluorescent signal for FACS. Nuclei were washed, resuspended in PBTB, and stored at 4°C until sorting.
Nuclei were filtered through a 40-μm cell strainer, and stained with DAPI for 30 min or longer before sorting. All sorting experiments were performed on either a BD FACSAria II or a BD FACSAria Fusion flow cytometer at a flow rate of ~20,000 events/sec. Singlets were gated using both forward scatter and side scatter. Nuclei from primary spermatocytes (4C) were gated based on DNA content deduced from the DAPI signal, and sorted into populations of interest based on fluorochrome intensity into collection tubes containing PBTB. Sorted nuclei were collected by centrifugation. Nuclei were examined under microscope and counted with a hemocytometer. Finally, excess buffer was removed and nuclei pellets were stored at −80°C.

tag: genotype

tag: antibody


title: STL002.PA2.ADS371

description:
PA.pp.all.bz2
PA.po.all.bz2

source_name: Human Pancreas

value: Pancreas


title: STL003.PO3.ADS230

description:
PO.pp.all.bz2
PO.po.all.bz2

source_name: Human Psoas

value: Psoas


title: H4K8ac  ChIP-Seq SCP3 positive H1t negative nuclei

value: Anti-H4K8ac Abcam (ab15823)


title: 1796

value: batch 2


title: H4K20me3  ChIP-Seq SCP3 positive H1t negative nuclei

value: Anti-H4K20me3 Millipore (07-463)


title: STL002.PA2.ADS129

description:
PA.pp.all.bz2
PA.po.all.bz2


title: 4492

value: male


title: 558


title: 1738


title: 2091


title: STL002.OV2.ADS372

description:
OV.pp.all.bz2
OV.po.all.bz2

source_name: Human Ovary

value: Ovary


title: H4K12ac  ChIP-Seq SCP3 positive H1t negative nuclei

value: wild-type

value: adult

value: Anti-H4K12ac Active motif (39928)


title: STL001.RV1.ADS130

description:
RV.pp.all.bz2
RV.po.all.bz2

value: Right Ventricle


title: H3K9me3  ChIP-Seq SCP3 positive H1t negative nuclei

value: whole testis

value: Anti-H3K9me3 Active motif (39766)


title: 1686


title: STL003.RA3.ADS221

description:
RA.pp.all.bz2
RA.po.all.bz2

value: Right Atrium


title: H1.MSC.rep2.ADS255

description:
msc.pp.all.bz2
msc.po.all.bz2

source_name: Human Mesenchymal Stem Cell

value: Mesenchymal Stem Cell


title: H3K9me2  ChIP-Seq SCP3 positive H1t negative nuclei

value: Anti-H3K9me2 Abcam (ab1220)


title: 1955


title: H3K9ac  ChIP-Seq SCP3 positive H1t negative nuclei

value: Anti-H3K9ac Active motif (39918)


title: 1655


title: H1.MSC.rep2.ADS254

description:
msc.pp.all.bz2
msc.po.all.bz2


title: H3K79me3  ChIP-Seq SCP3 positive H1t negative nuclei

value: Anti-H3K79me3 Abcam (ab2621)


title: 1637


title: H3K79me1  ChIP-Seq SCP3 positive H1t negative nuclei

value: Anti-H3K79me1 Abcam (ab2886)


title: H1.MSC.rep1.ADS225

description:
msc.pp.all.bz2
msc.po.all.bz2


title: H1.MSC.rep1.ADS215

description:
msc.pp.all.bz2
msc.po.all.bz2


title: H3K4me1  ChIP-Seq SCP3 positive H1t negative nuclei

value: Anti-H3K4me1 Abcam (ab8895)


title: 1496

value: batch 2


title: H3K4ac  ChIP-Seq SCP3 positive H1t negative nuclei

value: Anti-H3K4ac Abcam (ab176799)


title: H3K4me2  ChIP-Seq SCP3 positive H1t negative nuclei

value: male

value: Anti-H3K4me2 Active motif (39914)


title: 1454


title: 1447


title: 1436


title: H3K27ac  ChIP-Seq SCP3 positive H1t negative nuclei

value: wild-type

value: Anti-H3K27ac Abcam (ab177178)


title: 5974


title: H3K27me1  ChIP-Seq SCP3 positive H1t negative nuclei

value: adult

value: Anti-H3K27me1 Millipore (07-448)


title: 1578


title: H3K27me3  ChIP-Seq SCP3 positive H1t negative nuclei

value: whole testis

value: Anti-H3K27me3 Millipore (07-449)


title: 1344


title: H3K36me3  ChIP-Seq SCP3 positive H1t negative nuclei

value: Anti-H3K36me3 Active motif (61102)


title: 1301


title: H3  ChIP-Seq SCP3 positive H1t negative nuclei

value: Anti-H3 Abcam (ab1791)


title: 1280


title: abK4me ChIP-Seq Diplotene nuclei (Expt R2)

source_name: abK4me ChIP-Seq Diplotene nuclei

value: Diplotene nuclei

value: Anti-H3K4me2/3 Abcam (ab8580)


title: H3K4me3 ChIP-Seq Diplotene nuclei (Expt R2)

source_name: H3K4me3 ChIP-Seq Diplotene nuclei

value: Anti-H3K4me3 Epicypher 13-0028


title: input ChIP-Seq Diplotene nuclei (Expt R1)

source_name: input ChIP-Seq Diplotene nuclei


title: 1269


title: input ChIP-Seq Diplotene nuclei (Expt R2)


title: 1530


title: H3K9Ac ChIP-Seq Diplotene nuclei (Expt R1)

source_name: H3K9Ac ChIP-Seq Diplotene nuclei

value: Anti-H3K4me3 Active Motif 39918


title: 5295


title: 1211


title: STL002.LG2.ADS343

description:
LG.pp.all.bz2
LG.po.all.bz2

source_name: Human Lung

value: Lung


title: H3K4me3 ChIP-Seq Diplotene nuclei (Expt R1)


title: input ChIP-Seq Late Pachytene nuclei (Expt R2)

source_name: input ChIP-Seq Late Pachytene nuclei

value: Late Pachytene nuclei


title: STL002.LG2.ADS204

description:
LG.pp.all.bz2
LG.po.all.bz2


title: 1058

source_name: subcutaneous adipose tissue

value: batch 2


title: abK4me ChIP-Seq Late Pachytene nuclei (Expt R2)

source_name: abK4me ChIP-Seq Late Pachytene nuclei

value: male


title: STL001.LG1.ADS219

description:
LG.pp.all.bz2
LG.po.all.bz2


title: 1018


title: 1049


title: STL001.LG1.ADS201

description:
LG.pp.all.bz2
LG.po.all.bz2


title: input ChIP-Seq Late Pachytene nuclei (Expt R1)


title: H3K9Ac ChIP-Seq Late Pachytene nuclei (Expt R1)

source_name: H3K9Ac ChIP-Seq Late Pachytene nuclei


title: STL001.LG1.ADS198

description:
LG.pp.all.bz2
LG.po.all.bz2


title: 998


title: 1082


title: STL011.LI11.ADS394

description:
LI.pp.all.bz2
LI.po.all.bz2

source_name: Human Liver

value: Liver


title: 854


title: 582


title: input ChIP-Seq Early Pachytene nuclei (Expt R2)

source_name: input ChIP-Seq Early Pachytene nuclei

value: wild-type

value: Early Pachytene nuclei


title: STL001.LG1.ADS384

description:
LG.pp.all.bz2
LG.po.all.bz2


title: STL003.LV3.ADS463

description:
LV.pp.all.bz2
LV.po.all.bz2

source_name: Human Left Ventricle

value: Left Ventricle


title: 847


title: STL003.LV3.ADS461

description:
LV.pp.all.bz2
LV.po.all.bz2


title: abK4me ChIP-Seq Early Pachytene nuclei (Expt R2)

source_name: abK4me ChIP-Seq Early Pachytene nuclei

value: adult


title: H3K4me3 ChIP-Seq Late Pachytene nuclei (Expt R1)

source_name: H3K4me3 ChIP-Seq Late Pachytene nuclei

value: whole testis


title: 993


title: H3K4me3 ChIP-Seq Early Pachytene nuclei (Expt R2)

source_name: H3K4me3 ChIP-Seq Early Pachytene nuclei


title: STL003.LV3.ADS125

description:
LV.pp.all.bz2
LV.po.all.bz2


title: input ChIP-Seq Early Pachytene nuclei (Expt R1)


title: 799


title: H3K4me3 ChIP-Seq Late Pachytene nuclei (Expt R2)


title: case3

source_name: A498 cell line

tag: cell line


title: STL001.LV1.ADS126

description:
LV.pp.all.bz2
LV.po.all.bz2


title: H3K9Ac ChIP-Seq Early Pachytene nuclei (Expt R1)

source_name: H3K9Ac ChIP-Seq Early Pachytene nuclei


title: 787


title: 758


title: H3K4me3 ChIP-Seq Early Pachytene nuclei (Expt R1)


title: H1.ADS389

description:
H1.pp.all.bz2
H1.po.all.bz2

source_name: Human Human ES Cells

value: Human ES Cells


title: input ChIP-Seq Zygotene nuclei (Expt R2)

source_name: input ChIP-Seq Zygotene nuclei

value: Zygotene nuclei


title: H1.ADS127

description:
H1.pp.all.bz2
H1.po.all.bz2


title: 718


title: 635


title: abK4me ChIP-Seq Zygotene nuclei (Expt R2)

source_name: abK4me ChIP-Seq Zygotene nuclei


title: 634


title: 618


title: hg19.HC.ADS205

description:
HC.pp.all.bz2
HC.po.all.bz2

source_name: Human Hippocampus

value: Hippocampus


title: 602


title: 1944


title: input ChIP-Seq Zygotene nuclei (Expt R1)


title: H3K9Ac ChIP-Seq Zygotene nuclei (Expt R1)

source_name: H3K9Ac ChIP-Seq Zygotene nuclei


title: hg19.HC.ADS395

description:
HC.pp.all.bz2
HC.po.all.bz2


title: STL003.GA3.ADS229

description:
GA.pp.all.bz2
GA.po.all.bz2

source_name: Human Gastric

value: Gastric


title: 579

source_name: subcutaneous adipose tissue


title: 553


title: input ChIP-Seq Leptotene nuclei (Expt R2)

source_name: input ChIP-Seq Leptotene nuclei

value: Leptotene nuclei


title: H1.ADS393

description:
H1.pp.all.bz2
H1.po.all.bz2


title: STL003.GA3.ADS199

description:
GA.pp.all.bz2
GA.po.all.bz2


title: 539


title: 535


title: IMR90.ADS200

description:
imr90.pp.all.bz2
imr90.po.all.bz2

source_name: Human Fibroblasts

value: Fibroblasts


title: abK4me ChIP-Seq Leptotene nuclei (Expt R2)

source_name: abK4me ChIP-Seq Leptotene nuclei

value: Anti-H3K4me2/3 Abcam (ab8580)


title: input ChIP-Seq Leptotene nuclei (Expt R1)


title: H3K4me3 ChIP-Seq Zygotene nuclei (Expt R2)

source_name: H3K4me3 ChIP-Seq Zygotene nuclei

value: Anti-H3K4me3 Epicypher 13-0028


title: H3K9Ac ChIP-Seq Leptotene nuclei (Expt R1)

source_name: H3K9Ac ChIP-Seq Leptotene nuclei

value: Anti-H3K4me3 Active Motif 39918


title: hg19.HC.ADS214

description:
HC.pp.all.bz2
HC.po.all.bz2


title: 449

value: batch 2


title: IMR90.ADS115

description:
imr90.pp.all.bz2
imr90.po.all.bz2


title: 441

value: male


title: H3K4me3 ChIP-Seq Leptotene nuclei (Expt R1)

source_name: H3K4me3 ChIP-Seq Leptotene nuclei


title: 1234


title: STL002.EG2.ADS359

description:
EG.pp.all.bz2
EG.po.all.bz2

source_name: Human Esophagus

value: Esophagus


title: STL002.FT2.ADS370

description:
FT.pp.all.bz2
FT.po.all.bz2

source_name: Human Fat

value: Fat


title: H3K4me3 ChIP-Seq Leptotene nuclei (Expt R2)

value: wild-type


title: hg19.DLPFC.ADS376

description:
DLPFC.pp.all.bz2
DLPFC.po.all.bz2

source_name: Human Dorsolateral Prefrontal Cortex (DLPFC)

value: Dorsolateral Prefrontal Cortex (DLPFC)


title: ∆ctx rep2 (cecum)

source_name: Cecum of 3-day old infant rabbits infected with V. cholerae

tag: breed (host)

value: New Zealand rabbit

tag: tissue (host)

value: Cecum

value: {delta}ctx bacteria

tag: time

value: one-day post-intragastric infection


title: IMR90.ADS91

description:
imr90.pp.all.bz2
imr90.po.all.bz2


title: 379

tag: Sex

tag: sequencing batch


title: 10664

value: batch 1


title: hg19.DLPFC.ADS207

description:
DLPFC.pp.all.bz2
DLPFC.po.all.bz2


title: 472


title: 495


title: STL001.BL1.ADS380

description:
BL.pp.all.bz2
BL.po.all.bz2

source_name: Human Bladder

value: Bladder


title: 5550


title: STL001.BL1.ADS220

description:
BL.pp.all.bz2
BL.po.all.bz2


title: 420


title: STL001.BL1.ADS202

description:
BL.pp.all.bz2
BL.po.all.bz2


title: STL001.BL1.ADS231

description:
BL.pp.all.bz2
BL.po.all.bz2


title: 2881


title: 10385


title: STL003.AO3.ADS386

description:
AO.pp.all.bz2
AO.po.all.bz2

source_name: Human Aorta

value: Aorta


title: 2554


title: STL003.AO3.ADS203

description:
AO.pp.all.bz2
AO.po.all.bz2


title: 1971


title: 5453