title: SI-NET-seq K562 dTAG7 2h R1

city: Innsbruck

first: Univ.-Prof. Dr. Andreas

last: Mayer

address: Innrain 80/82

institute: Biocenter, Medical University Innsbruck

description: 10 nt random molecular barcodes

data_processing:
Library strategy: NET-seq
Data processing of SI-NET-seq data sets was performed similar as described in Mayer et al., 2015.
Pre-processing: The six or ten 5’ end nucleotides corresponding to the molecular barcode, are trimmed from the reads, but remain associated with the read using a custom python script.
Mapping: Reads are aligned using the STAR aligner (v2.5.3a) (Dobin et al., 2013) with the following parameters : --clip3pAdapterSeq ATCTCGTATGCCGTCTTCTGCTTG --clip3pAdapterMMp 0.21 --clip3pAfterAdapterNbases 1 –outFilterMultimapNmax 1000000000 –outSJfilterOverhangMin 3 1 1 1 –outSJfilterDistToOtherSJmin 0 0 0 0 –alignIntronMin 11 –alignEndsType EndToEnd. The reference genome consisted of a joined reference genome which we assembled from human (GRCh38.p12) and mouse (GRCm38.p6) reference genomes, with additional sequences containing the rDNA sequences (genBank U13369.1, genBank U67616.1), and the sequence of processed tRNA (with the additional CCA at their end, and intron removed, when present). The alignment was performed with the GENCODE annotation v28.
Filtering: We separated reads mapping to the mouse genome for normalization. We removed putative contaminations such as reverse transcription mispriming events (barcode sequences correspond exactly to the genomic sequence adjacent to the aligned read), PCR duplicates (reads that align to the same genomic position and contain identical barcode) and splicing intermediates (exact single bp 3’ ends of introns and exons).
Normalization: We extracted the position corresponding to the 5’ end of the sequencing read (after removal of the barcode), which corresponds to the 3’ end of the nascent RNA fragment, using the HTSeq package (Anders et al., 2014). The Reference-adjusted Reads Per Million (RRPM) were calculated as described by (Orlando et al., 2014) using the number of reads mapping to the mouse genome as reference.
Genome_build: Human (hg38/GRCh38) and the spiked-in mouse (mm10/GRCm38)
Supplementary_files_format_and_content: Bedgraph files; Scores represent Reference-adjusted Reads Per Million (RRPM).

source_name: bone marrow, CML

growth_protocol: K562 dTAG-BRD4 and MOLT4 cells were cultured in RPMI containing 10% FBS, 5% penicillin-streptomycin. NIH3T3 cells, which were used as spike-ins, were grown in DMEM containing 10% FBS , 5% penicillin-streptomycin. K562 dTAG-BRD4 and MOLT4 cells were seeded at 5 x 10^5 cells/ml, NIH/3T3 were seeded at 2 x 10^6 cells/T75 flask every two days.

extract_protocol:
Nascent RNA was extracted as described by Mayer and Churchman (2016) with the following modification: After treatment of MOLT4 or K562 dTAG-BRD4, NIH3T3 cells were added in a ratio of 1:6.
Libraries were constructed as described by Mayer and Churchman (2016). In case of the libraries on K562 dTAG-BRD4,  the following modifications were implemented: After purification of the nascent RNA, a DNA linker containing a random decamer sequence was ligated to the 3'-end of the transcripts. Ligated RNA was fragmented by incubation with NEBNext RNA fragmentation solution at 95°C for 10 min. To improve read mapping and take the increased linker length into account, all size selection steps were adapted.

treatment_protocol: K562 dTAG-BRD4 cells at a concentration of 1 x 10^6/ml were treated with 100 nM dBET6, 500 nM dTAG7 or DMSO for 40, 50, 60 or 120 min.

value: K562 dTAG-BRD4

value: BRD4 degradation

tag: treatment time

tag: molecule subtype

value: nascent RNA


title: SI-NET-seq K562 dTAG7 2h R2

status: Public on Jul 30 2021

type: SRA

department: Institute for Genomics and RNomics

country: Austria

description: 10 nt random molecular barcodes

library_source: transcriptomic

molecule: total RNA

organism: Homo sapiens

tag: cell line

tag: treatment


title: Wi38 P1

city: Berlin

first: Clemens

last: Schmitt

address: Augustenburger Platz 1

department: Hematology, Oncology, Tumorimmunology

country: Germany

institute: Charite

description:
control Wi38 proliferating
AS-401822-LR-46699
OIS_norm_counts

data_processing:
Transcript quantification using salmon (Patro et al.)
Transcript import using tximeta (Love et al.) or tximport packages (Soneson et al.).
Genome_build: hg19
Supplementary_files_format_and_content: Matrix table with normalized transcript counts for every gene and every sample

growth_protocol: A549 and Wi38 cells were cultivated in DMEM supplemented with 10% FBS and Pen-Strep antibiotics; THP-1 cells were cultivated in RPMI-1640 supplemented with 10% FBS and Pen-Strep antibiotics

extract_protocol:
Total RNA was extracted using Qiagen RNeasy Plus Mini Kit
Libraries were prepared by BGI or DKFZ sequencing core facility according to Illumina's TruSeq RNA kit (RS-122-2001) instructions.

treatment_protocol: Cells were infected with virus or cultivated in conditioned medium for 5 days (THP-1: 3 days) and cell pellets were collected for RNA extraction. THP1 VIS samples were split into CD86+ and CD86- fractions using surface labelung with a CD86-biotin antibody and anti-biotin Microbeads from Miltenyi, following the manufacturer´s instructions.

tag: condition


title: Wi38 P2

description:
control Wi38 proliferating
AS-401823-LR-46699
OIS_norm_counts


title: Med16-AID, TBP-ChEC (IAA) rep-1

city: Bloomington

first: Gabriel

middle: E

last: Zentner

state: IN

address: 915 E 3rd St

department: Biology

country: USA

institute: Indiana University

library_source: genomic

data_processing:
Library strategy: ChEC-seq
Alignment with Bowtie2 (default settings plus "--no-unal --dovetail –no-discordant --no-mixed")
samtools view (default settings)
samtools sort (default settings)
samtools index (default settings)
Deeptools bamCoverage (default settings plus "--ignoreDuplicates -bs 10 --normalizeUsing CPM" )
Genome_build: sacCer3
Supplementary_files_format_and_content: bigWig

molecule: genomic DNA

organism: Saccharomyces cerevisiae

growth_protocol: Cells were grown at 30℃ in YPD to an OD600 of 0.7-1.0

extract_protocol:
ChEC was performed as described in Donczew et al. 2020, PMID: 31913117
NEB Next Ultra II DNA Library Prep Kit for Illumina

treatment_protocol: For the Med16-AID strains, 3-IAA treated samples were treated with 3-IAA in DMSO at a final concentration of 0.5mM for 30 minutes. DMSO-treated samples were treated with DMSO (1:2000 by culture volume) for 30 minutes

tag: chec tag

tag: auxin-inducible degron (aid) tag

tag: background


title: stool small RNA-Seq VOV68

city: Orbassano

first: Giulio

last: Ferrero

state: TO

address: Regione Gonzole, 10

department: Dept. Clinical and Biological Sciences

country: Italy

institute: University of Turin

data_processing:
Fastq files were quality-controlled with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Reads shorter than 14 nt were discarded.
The QC-passed reads were clipped from the adapter sequences using Cutadapt v1.10 by imposing a maximum error rate in terms of mismatches, insertions, and deletions equal to 0.15.
Trimmed reads were mapped against a miRNA reference based on miRBase v22 annotations using the small RNA-Seq module of Docker4Seq. The alignment was performed using BWA algorithm v0.7.127 with the default settings
The human miRNAs were annotated and quantified using two methods called the "knowledge-based" and "position-based". miRNAs whose assigned arms were derived from the "position-based" methodology were indicated in italics. The results generated by the annotation and quantification methods were merged into a unique mature miRNA count matrix. In the case of mature miRNAs processed from different precursors but characterized by the same mature sequence, the read counts were summed.
Genome_build: hg38
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

source_name: Stool

extract_protocol:
RNA was extracted using the Stool Total RNA Purification Kit (Norgen Biotek Corp).
Small RNA (sRNA) transcripts were converted into barcoded cDNA libraries. Library preparation was performed with the NEBNext Multiplex Small RNA Library Prep Set for Illumina (Protocol E7330, New England BioLabs Inc., USA). The obtained libraries were subjected to the Illumina sequencing pipeline, passing through clonal cluster generation on a single-read flow cell (Illumina Inc., USA) by bridge amplification on the cBot (TruSeq SR Cluster Kit v3-cBOT-HS, Illumina Inc., USA) and 50 cycles sequencing-by-synthesis on the HiSeq 2000 (Illumina Inc., USA).

tag: diet

value: Vegetarian


title: stool small RNA-Seq VOV67


title: SI-NET-seq K562 dBET6 2h R1

description: 10 nt random molecular barcodes

value: pan-BET degradation


title: stool small RNA-Seq VOV69


title: stool small RNA-Seq VOV72


title: stool small RNA-Seq VOV61


title: SI-NET-seq K562 DMSO 2h R2

description: 10 nt random molecular barcodes

value: control


title: stool small RNA-Seq VOV60


title: OC9.3

city: Baltimore

first: Carrie

last: McCracken

state: MD

address: 670 W. Baltimore St

department: Institute for Genome Sciences

institute: Institute of Genome

data_processing:
Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software
The STAR aligner uses a splice aligner that detects splice junctions and incorporates them to help align the entire read sequences. BAM files were generated as a result of this step. Unique gene hit counts were calculated by using featureCounts from the Subread package v.1.5.2.
Using DESeq2, a comparison of gene expression between the groups of samples was performed. The Wald test was used to generate p-values and Log2 fold changes.
Genome_build: The trimmed reads were mapped to the Homo sapiens GRCh38 reference genome available on ENSEMBL using the STAR aligner v.2.5.2b.
Supplementary_files_format_and_content: tab-delimted text files include counts values for each sample

source_name: Oseochondroma

growth_protocol: Cartilage caps were microdissected from human osteochondromas obtained at surgery, miced into 3-5 mm cubic and cultured inside a diffusion chamber

extract_protocol:
Osteochondroma explants were washed with PBS, stored in RNAlator and kept at -80C. The explants were placed on clean Kimwipe to absorb excess RNAlater solution and then were snap-frozen in liquid nitrogen. Frozen tissue fragments were placed on pre-chilled carbon steel block and crashed into powder by the steel hammer. We then purified total RNA using Qiagen RNeasy fibrous tissue mini kit
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Osteochondroma explants were treated with NRX202467 or Palovarotene at 50-300nM.

tag: patient

value: Patient #9

tag: treated drug

tag: concentration


title: stool small RNA-Seq VOV58

value: Vegan


title: stool small RNA-Seq VOV56


title: SI-NET-seq K562 DMSO 2h R1

description: 10 nt random molecular barcodes


title: stool small RNA-Seq VOV55


title: Med15-AID, +IAA, rep-3

data_processing:
Library strategy: nsRNA-Seq
Alignment with STAR v2.6.1a
Quantification of reads per gene with STAR --quant mode
Differential expression analysis was performed with DESeq2. Size factors were determined by estimateSizeFactors on the count matrix of S.pombe reads for all of the samples. These size factors were then used for the S. cerevisiae libraries using "sizeFactors="
Genome_build: sacCer3
Supplementary_files_format_and_content: raw counts per gene

organism: Saccharomyces cerevisiae W303

growth_protocol: Grown at 30C in YPD

extract_protocol:
Cells were labelled with 4tU for 6 minutes. Total RNA was extracted using the Masterpure Yeast RNA extraction kit (Lucigen MPY03100) as per the manufacturer’s protocol. Total RNA was subjected to biotinylation, pull-down and purification as previously described in Donczew et al, 2020 [PMID:31913117]. rRNA was depleted from the purified nascent RNA fraction using Terminator™ 5′-Phosphate-Dependent Exonuclease (Lucigen TER51020) as per the manufacturer’s protocol. rRNA-depleted nascent RNA was purified and concentrated using RNAClean XP clean beads (1.8:1 beads:sample ratio).
TruSeq Stranded Total RNA kit for Illumina

treatment_protocol: 3-IAA-treated samples were treated with 3-IAA in DMSO at a final concentration of 0.5mM for 30 minutes. DMSO-treated samples were treated with DMSO (1:2000 by culture volume) for 30 minutes.

tag: strain


title: Med15-AID, +DMSO, rep-3


title: stool small RNA-Seq VOV53


title: stool small RNA-Seq VOV64


title: stool small RNA-Seq VOV54


title: DASLV4_18

type: RNA

city: York

first: Matthew

middle: Anthony

last: Care

state: North Yorkshire

address: Wentworth Way

department: Department of Biology

country: United Kingdom

institute: The University of York

hyb_protocol: Standard Illumina protocol

scan_protocol: Arrays were scanned on a BeadArray reader

data_processing: Data were normalised using Lumi for R.

label: Biotin

source_name: Biopsy

label_protocol: Standard Illumina protocol

extract_protocol: Total RNA was extracted from 5mm paraffin sections using the Ambion RecoverAll kit standard protocol, with the exception of a 16-hour protease digestion. These were routinely collected tissue samples and no attempt was made to standardise fixation or processing protocols

value: DLBCL

value: Diffuse large B-cell lymphoma, NOS

tag: reagent

tag: microdissection

value: FALSE

value: GCB

tag: Sex

value: F

value: CHOP-R

value: Y

tag: Stage

value: III

tag: ldh

value: raised

tag: albumin

tag: hb

tag: lymphs

tag: wbc

value: Yes

value: TRUE


title: Med15-AID, +IAA, rep-2

tag: treatment


title: Med15-AID, +DMSO, rep-2

tag: auxin-inducible degron (aid) tag


title: stool small RNA-Seq VOV59


title: HiChIP-H3K27ac_day-0_rep-1

city: Stanford

first: Daniel

middle: Sunwook

last: Kim

state: CA

address: 269 Campus Drive, CCSR 2150

institute: Stanford School of Medicine

description:
protein enriched long range contact
FitHiChIP.d0_pooled.interactions_FitHiC_Q0.01_WashU.bed
ggr.linking.d0.overlap.idr_filt.interactions.txt.gz
ggr.linking.ALL.overlap.interactions.txt.gz

data_processing:
Library strategy: HiChIP
Library strategy: HiChIP
Sequencing reads were aligned and paired, filtered for long0range contacts, and removed of duplicates using the HiC-Pro pipeline (PMID: 26619908)
Filtered reads were processed with hichipper to call peaks
HiC-Pro valid interaction pairs and hichipper peaks were processed through FitHiChIP to call significant chromatin contacts
Significant contacts were called on each replicate as well as pooled data - significant contact is then kept if it was called in the pooled and significant in IDR framework
Genome_build: hg19
Supplementary_files_format_and_content: Significant interactions per day, filtered interactions per day, all interactions across timepoints (union)

source_name: Primary foreskin keratinocytes

growth_protocol: Human primary keratinocytes were cultured in 50/50 growth media and induced to differentiate for 6 days as previously describe (Zarnegar 2012)

extract_protocol:
Protein enrichment of 3C library
Protocol as described in Mumbach 2016

treatment_protocol: Crosslinked with 1% formaldehyde

tag: cell type

value: Primary foreskin keratinocytes

tag: timepoint

tag: tag


title: HiChIP-H3K27ac_day-0_rep-2


title: stool small RNA-Seq VOV51

tag: diet

value: Omnivorous


title: HiChIP-H3K27ac_day-6_rep-2

description:
protein enriched long range contact
FitHiChIP.d6_pooled.interactions_FitHiC_Q0.01_WashU.bed
ggr.linking.d6.overlap.idr_filt.interactions.txt.gz
ggr.linking.ALL.overlap.interactions.txt.gz


title: HiChIP-H3K27ac_day-3_rep-1

description:
protein enriched long range contact
FitHiChIP.d3_pooled.interactions_FitHiC_Q0.01_WashU.bed
ggr.linking.d3.overlap.idr_filt.interactions.txt.gz
ggr.linking.ALL.overlap.interactions.txt.gz


title: HiChIP-H3K27ac_day-6_rep-1


title: stool small RNA-Seq VOV52


title: stool small RNA-Seq VOV50


title: Mot1/Med16-AID, +DMSO, rep-3

value: Med16-3xV5-IAA7, Mot1-3xHA-IAA7


title: Med15-AID, +DMSO, rep-1


title: Mot1/Med16-AID, +IAA, rep-3


title: Med15-AID, +IAA, rep-1


title: stool small RNA-Seq VOV48

value: Vegetarian


title: Mot1/Med16-AID, +DMSO, rep-2


title: Mot1/Med16-AID, +IAA, rep-2


title: stool small RNA-Seq VOV63


title: stool small RNA-Seq VOV47


title: Mot1/Med16-AID, +IAA, rep-1


title: Mot1/Med16-AID, +DMSO, rep-1


title: Med15/Med16-AID, +IAA, rep-3

value: Med16-3V5-IAA7, Med15-3xHA-IAA7


title: stool small RNA-Seq VOV46


title: stool small RNA-Seq VOV45


title: Med15/Med16-AID, +IAA, rep-2


title: Med15/Med16-AID, +IAA, rep-1


title: Med15/Med16-AID, +DMSO, rep-3


title: Med15/Med16-AID, +DMSO, rep-1


title: stool small RNA-Seq VOV43

value: Vegan


title: med16-deletion, rep-2


title: med16-deletion, rep-1


title: stool small RNA-Seq VOV41


title: Med15/Med16-AID, +DMSO, rep-2


title: DLX1-KO-3

city: Bangalore

first: Genotypic

last: technology

state: Karnataka

address: 259, Apoorva 4th cross,80 feet Road,RMV 2ND STAGE

country: India

institute: Genotypic Technology

description:
CRISPR/Cas9 mediated DLX1 knockout
22RV1 DLX1-KO C-3 (Cy5) vs. 22RV1 SCR (Cy3)

hyb_protocol: Cy3 labeled samples and of Cy5 labeled samples were pooled. Fragmentation of labeled cRNA and hybridization were done using the Gene Expression Hybridization kit of Agilent (Part Number 5190-0404). Hybridization was carried out in Agilent’s Surehyb Chambers at 65º C for 16 hours. The hybridized slides were washed using Agilent Gene Expression wash buffers (Part No: 5188-5327)

scan_protocol: Scanned using Agilent Scanner (Agilent Technologies G2600D ).

data_processing: The Lowess Normalization was done using GeneSpring GX Software.

source_name: 22RV1 Scramble cells

label_protocol: The samples for Gene expression were labeled using Agilent Quick Amp Kit PLUS (Part number: 5190-0444). Each of the Control and test samples were reverse transcribed at 40°C and  converted to double stranded cDNA primed by oligodT tagged with a T7 polymerase promoter. The double stranded cDNA were used as template for cRNA synthesis. cRNA was generated by in vitro transcription. The dye Cy3 CTP (Agilent) for control samples and Cy5 CTP(Agilent) for test samples were incorporated during this step. The cDNA synthesis and in vitro transcription steps were carried out at 40°C. Labeled cRNA was cleaned up and quality assessed for yields and specific activity using Nanodrop.

growth_protocol: 22RV1 cells were co-transfected with pCas-Guide and donor vector. Transfected cells were split in 1:10 and cultured for 8 passages in  RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS), 0.5% Penicillin Streptomycin (Pen-Strep). Single cell clones with DLX1 gene knockout were selected using  Puromycin (1µg/mL).

extract_protocol: Total RNA was extracted from DLX1-KO and SCR control cells using Qiaigen miRNeasy mini Kit (Cat: 217004) according to the manufacturer protocol.

value: Prostate cancer cells

tag: cell line

tag: genotype

value: Scramble control

source_name: 22RV1 DLX1-KO cells


title: stool small RNA-Seq VOV44


title: DLX1-KO-1

description:
CRISPR/Cas9 mediated DLX1 knockout
22RV1 DLX1-KO C-1 (Cy5) vs. 22RV1 SCR (Cy3)


title: wildtype, rep-2

tag: strain


title: OSC28_GapMalat1 vs OSC28_GapLacZ_NORM

city: Biella

first: Paola

last: Ostano

address: via Malta 3

institute: Fondazione Edo ed Elvo Tempia

hyb_protocol: mRNA from 100 ng of totRNA was amplified, labeled with Cy3 or Cy5 and purified with columns. 300 ng of differentially labeled specimens were hybridized on the Agilent SurePrint G3 Human Gene Expression v3, 8x60K Microarrays (Agilent, Santa Clara, CA, USA).

scan_protocol: After hybridization, slides were washed and scanned using the G2505C Agilent scanner.

data_processing: Raw data elaboration was performed with the LIMMA (LInear Models for Microarray Analysis), within Bioconductor. Raw intensity values were background subtracted (method = normexp, offset = 50) and normalized using the loess method, for the within array normalization, and Aquantile, for the between array normalization. Results were represented in terms of log2Ratios for heatmaps and Ratios for dot-plots, where Ratios indicate the mean normalized fluorescence in Malat1-depleted samples respect to controls.

label_protocol: Gene expression profiling was carried out using the two-color labeling method by means of Low Input Quick Amp Labeling Kit (Agilent Technologies).

growth_protocol: Prostate cancer cells (C27IM, LNCaP, PC3 and PC3-AR) were cultured as described in Aiello et al 2016. PCa patients were enrolled at the Department of Urology, Università Cattolica del Sacro Cuore, Rome Italy (from October 2015 to February 2020) to perform prostatectomy. OSCs were generated and RNA extracted and processed as described in Aiello 2016. Protein extraction was performed as described in Bacci el al 2019.

extract_protocol: RNA was extracted from two independent biological replicates  of three PCa cell lines (C27IM, DU145, and PC3) and from three primary-tumor-derived OSCs with MALAT1 depletion compared to GapLacZ controls. RNA quality and quantity were assessed using Agilent 2100 bioanalyzer (Agilent Technologies) and NanoDrop ND-1000 spectro-photometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA), respectively

treatment_protocol: MALAT1 silencing was achieved by specific gapmers or LacZ control gapmers as described in Aiello et al 2016.

value: Malat1 depletion

value: control


title: wildtype, rep-1


title: stool small RNA-Seq VOV40


title: OSC11_GapMalat1 vs OSC11_GapLacZ_NORM


title: DLX1-KO-2

description:
CRISPR/Cas9 mediated DLX1 knockout
22RV1 DLX1-KO C-2 (Cy5) vs. 22RV1 SCR (Cy3)


title: stool small RNA-Seq VOV42


title: OSC12_GapMalat1 vs OSC12_GapLacZ_DS


title: OSC28_GapMalat1 vs OSC28_GapLacZ_DS


title: PC3_GapMalat1 vs PC3_GapLacZ_DS

value: PC3 prostate cancer cell line


title: HiChIP-H3K27ac_day-3_rep-2


title: Med16-AID, +DMSO, rep-2


title: wildtype, rep-3


title: stool small RNA-Seq VOV03


title: Med16-AID, +IAA, rep-3


title: Med16-AID, +DMSO, rep-1


title: OSC12_GapMalat1 vs OSC12_GapLacZ_NORM


title: PC3_GapMalat1 vs PC3_GapLacZ_NORM2


title: med16-deletion, rep-3


title: Med16-AID, +IAA, rep-2


title: stool small RNA-Seq VOV38


title: Med16-AID, +DMSO, rep-3


title: DU145_GapMalat1 vs DU145_GapLacZ_DS

value: DU145 prostate cancer cell line


title: stool small RNA-Seq VOV36


title: PC3_GapMalat1 vs PC3_GapLacZ_NORM


title: Med14-ChEC in Med16-AID, +DMSO, rep-2

data_processing:
Library strategy: ChEC-seq
Alignment with Bowtie2 (default settings plus "--no-unal --dovetail –no-discordant --no-mixed")
HOMER makeTagDirectory
HOMER makeUCSCfile ("-bigWig -norm 1e6")
Genome_build: sacCer3
Supplementary_files_format_and_content: bigWig

extract_protocol:
ChEC performed as described in Tourigny et al. 2018, PMID: 30275344
NEB Next Ultra II DNA Library Prep Kit for Illumina

tag: chec tag


title: Med5-ChEC in Med16-AID, +DMSO, rep-2


title: Med16-AID, +IAA, rep-1


title: Med5-ChEC in Med16-AID, +IAA, rep-2


title: YPG_7d_rep2

city: Hamilton

first: Evan

last: Shepherdson

state: Ontario

address: 1280 Main St W

country: Canada

institute: McMaster University

data_processing:
RNA-seq reads were aligned to the Streptomyces venezuelae genome GCA_001886595.1 using Bowtie2
Aligned reads were sorted using Samtools sort and a counts file was generated using HTseq-count
Counts files were processed using DESeq2 to generate a table of normalized transcript abundance measurements and to analyze statistical changes in expression between sample conditions
Genome_build: GCA_001886595.1
Supplementary_files_format_and_content: Matrix table with normalized transcript abundance for every gene and every sample in .csv file format

organism: Streptomyces venezuelae

source_name: Bacterial cells

growth_protocol: 10 uL of a Streptomyces venezuelae overnight culture were spotted to solid growth medium and incubated at 30 degrees Celsius. After specified incubation times, total RNA was extracted from cell lysates.

extract_protocol:
Lysates were generated by vortexing bacterial cells with glass beads in a guanidinium thiocyanate saturated lysis buffer. Total RNA was extracted with acidified phenol:chloroform
rRNA was depleted from total RNA samples using a Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina. Libraries were constructed using an NEBNext Ultra II Direcitonal RNA Library Prep Kit for Illumina.

tag: growth medium

value: Yeast extract - peptone - glycerol (YPG)

tag: incubation time

value: 7 days

tag: sequencing platform

value: MiSeq


title: Med2-ChEC in Med16-AID, +IAA, rep-1


title: stool small RNA-Seq VOV35


title: Med2-ChEC in Med16-AID, +IAA, rep-2


title: Med2-ChEC in Med16-AID, +DMSO, rep-2


title: YPG_7d_rep1


title: SI-NET-seq K562 dBET6 2h R2

description: 10 nt random molecular barcodes

value: K562 dTAG-BRD4

value: pan-BET degradation

tag: treatment time

tag: molecule subtype

value: nascent RNA


title: Med2-ChEC in Med16-AID, +DMSO, rep-1


title: Med14-ChEC in Med16-AID, +IAA, rep-2


title: YPG_4d_rep1

value: 4 days


title: Med14-ChEC in Med16-AID, +DMSO, rep-1


title: stool small RNA-Seq VOV33

value: Omnivorous


title: DU145_GapMalat1 vs DU145_GapLacZ_NORM


title: YPG_2d_rep2

value: 2 days


title: stool small RNA-Seq VOV32

value: Vegetarian


title: C27IM_GapMalat1 vs C27IM_GapLacZ_NORM

value: C27IM prostate cancer cell line


title: Med5-ChEC in Med16-AID, +IAA, rep-1


title: Med5-ChEC in Med16-AID, +DMSO, rep-1


title: MED9 in WT, rep-3


title: C27IM_GapMalat1 vs C27IM_GapLacZ_DS


title: stool small RNA-Seq VOV34


title: stool small RNA-Seq VOV37


title: YPG_2d_rep1


title: KO_IL4_6

city: Cambridge

first: Brian

middle: Yee Hong

last: Lam

address: Box 289 Addenbookes Hospital, Hills Road

department: Metabolic Research Laboratories

institute: University of Cambridge

description:
IL-4 treated Bone Marrow derived Macrophages from LysM+/Cre SCAPfl/fl mice
SLX-14429.A027

data_processing:
Fastq reads were mapped with TopHat 2.0.11 to  mouse GRCm38 Ensembl V54
Counts were generated using htseq-count 0.6.1p1 on the reverse strand
Genome_build: GRCm38
Supplementary_files_format_and_content: raw_count.tsv in tab-delimiited format

organism: Mus musculus

source_name: Bone Marrow derived Macrophages

growth_protocol: BMDM were differentiated in RPMI with 10% heat-inactivated FBS 20% L929 cells media and 1% PS. After 1 week, BMDM were seeded into a 24 well plate at 250,000 cells per well in PMI with 10% heat-inactivated FBS 20% L929 cells media and 1% PS

extract_protocol:
Qiagen Rneasy
Illumina TruSeq Stranded mRNA Library Prep

treatment_protocol: 24h after plating, cells were treated or not with IL4 (10ng/ml) for 24h. Plates were rinse once with PBS and snap-frozen on dry ice.

value: Bone Marrow derived Macrophages

value: LysM+/Cre SCAPfl/fl

tag: batch

value: Batch 2


title: YPG_4d_rep2


title: stool small RNA-Seq VOV02


title: MED9 in WT, rep-1


title: stool small RNA-Seq VOV28


title: MED9 in  med16-deletion, rep-1


title: MED8 in WT, rep-3


title: KO_IL4_5

description:
IL-4 treated Bone Marrow derived Macrophages from LysM+/Cre SCAPfl/fl mice
SLX-14429.A025


title: MED8 in WT, rep-1


title: MED9 in  med16-deletion, rep-3


title: stool small RNA-Seq VOV29

value: Vegan


title: MED9 in  med16-deletion, rep-2


title: MED9 in WT, rep-2


title: MED8 in  med16-deletion, rep-4


title: MED8 in  med16-deletion, rep-3


title: MED8 in  med16-deletion, rep-2


title: stool small RNA-Seq VOV24


title: KO_IL4_2

description:
IL-4 treated Bone Marrow derived Macrophages from LysM+/Cre SCAPfl/fl mice
SLX-14429.A010

value: Batch 1


title: KO_IL4_4

description:
IL-4 treated Bone Marrow derived Macrophages from LysM+/Cre SCAPfl/fl mice
SLX-14429.A023


title: stool small RNA-Seq VOV25


title: Med16-AID, TBP-ChEC (DMSO) rep-1

tag: background


title: MED8 in  med16-deletion, rep-1

tag: treatment


title: MED5 in WT, rep-3

tag: auxin-inducible degron (aid) tag


title: stool small RNA-Seq VOV23


title: KO_IL4_1

description:
IL-4 treated Bone Marrow derived Macrophages from LysM+/Cre SCAPfl/fl mice
SLX-14429.A007

tag: cell type


title: stool small RNA-Seq VOV26


title: MED5 in WT, rep-2


title: MED5 in WT, rep-1


title: KO_IL4_3

description:
IL-4 treated Bone Marrow derived Macrophages from LysM+/Cre SCAPfl/fl mice
SLX-14429.A011


title: Med14-ChEC in Med16-AID, +IAA, rep-1


title: MED5 in  med16-deletion, rep-3


title: WT_IL4_4

description:
IL-4 treated Bone Marrow derived Macrophages from LysM+/+ SCAPfl/fl mice
SLX-14429.A020

value: LysM+/+ SCAPfl/fl


title: WT_IL4_5

description:
IL-4 treated Bone Marrow derived Macrophages from LysM+/+ SCAPfl/fl mice
SLX-14429.A021


title: WT_IL4_3

description:
IL-4 treated Bone Marrow derived Macrophages from LysM+/+ SCAPfl/fl mice
SLX-14429.A012


title: MED5 in  med16-deletion, rep-2


title: MED3 in WT, rep-3


title: MED5 in  med16-deletion, rep-1


title: stool small RNA-Seq VOV20


title: MED3 in  med16-deletion, rep-3


title: WT_IL4_6

description:
IL-4 treated Bone Marrow derived Macrophages from LysM+/+ SCAPfl/fl mice
SLX-14429.A022


title: stool small RNA-Seq VOV21


title: MED3 in WT, rep-2


title: MED3 in  med16-deletion, rep-2


title: WT_IL4_2

description:
IL-4 treated Bone Marrow derived Macrophages from LysM+/+ SCAPfl/fl mice
SLX-14429.A009


title: MED3 in  med16-deletion, rep-1


title: stool small RNA-Seq VOV16


title: MED2 in WT, rep-3


title: stool small RNA-Seq VOV15


title: MED2 in WT, rep-2


title: KO_CTR_6

description:
Control Bone Marrow derived Macrophages from LysM+/Cre SCAPfl/fl mice
SLX-14429.A019

value: Control


title: stool small RNA-Seq VOV14


title: MED2 in  med16-deletion, rep-2


title: stool small RNA-Seq VOV144


title: MED2 in  med16-deletion, rep-1


title: KO_CTR_5

description:
Control Bone Marrow derived Macrophages from LysM+/Cre SCAPfl/fl mice
SLX-14429.A018


title: MED16 in WT, rep-3


title: stool small RNA-Seq VOV141


title: KO_CTR_3

description:
Control Bone Marrow derived Macrophages from LysM+/Cre SCAPfl/fl mice
SLX-14429.A005


title: MED2 in WT, rep-1


title: KO_CTR_4

description:
Control Bone Marrow derived Macrophages from LysM+/Cre SCAPfl/fl mice
SLX-14429.A016


title: MED16 in WT, rep-1


title: MED2 in  med16-deletion, rep-3


title: KO_CTR_2

description:
Control Bone Marrow derived Macrophages from LysM+/Cre SCAPfl/fl mice
SLX-14429.A004


title: MED15 in WT, rep-2


title: MED15 in WT, rep-3


title: stool small RNA-Seq VOV140


title: MED15 in WT, rep-1


title: MED15 in  med16-deletion, rep-3


title: KO_CTR_1

description:
Control Bone Marrow derived Macrophages from LysM+/Cre SCAPfl/fl mice
SLX-14429.A001


title: MED15 in  med16-deletion, rep-2


title: stool small RNA-Seq VOV139


title: WT_IL4_1

description:
IL-4 treated Bone Marrow derived Macrophages from LysM+/+ SCAPfl/fl mice
SLX-14429.A008


title: WT_CTR_6

description:
Control Bone Marrow derived Macrophages from LysM+/+ SCAPfl/fl mice
SLX-14429.A015


title: MED15 in  med16-deletion, rep-1


title: MED14 in WT, rep-3


title: stool small RNA-Seq VOV137


title: MED14 in WT, rep-2


title: WT_CTR_5

description:
Control Bone Marrow derived Macrophages from LysM+/+ SCAPfl/fl mice
SLX-14429.A014


title: MED16 in WT, rep-2


title: MED14 in  med16-deletion, rep-2


title: MED14 in  med16-deletion, rep-3


title: WT_CTR_4

description:
Control Bone Marrow derived Macrophages from LysM+/+ SCAPfl/fl mice
SLX-14429.A013


title: stool small RNA-Seq VOV136


title: MED14 in  med16-deletion, rep-1


title: stool small RNA-Seq VOV135


title: MED13 in WT, rep-1.1


title: WT_CTR_3

description:
Control Bone Marrow derived Macrophages from LysM+/+ SCAPfl/fl mice
SLX-14429.A006


title: WT_CTR_2

description:
Control Bone Marrow derived Macrophages from LysM+/+ SCAPfl/fl mice
SLX-14429.A003


title: MED13 in  med16-deletion, rep-2


title: WT_CTR_1

description:
Control Bone Marrow derived Macrophages from LysM+/+ SCAPfl/fl mice
SLX-14429.A002


title: MED13 in  med16-deletion, rep-3


title: MED13 in  med16-deletion, rep-1


title: stool small RNA-Seq VOV133


title: m6_div6: Bcell-Div-CrePos_4_d6_RNAseq

city: Atlanta

first: Chris

last: Scharer

state: GA

address: 1510 Clifton Rd, Suite 3086A

department: Microbiology and Immunology

institute: Emory University

data_processing:
Raw sequencing reads were mapped to the mm10 using STAR and the UCSC KnownGene reference transcriptome.
Reads mapping to exons were summarized for each unique ENTREZ gene using the GenomicRanges package in R/Bioconductor and normalized to reads per kilobase per million (RPKM).
Genome_build: mm10
Supplementary_files_format_and_content: Annotation file containing RPKM normalized read counts for all detected genes.

molecule: polyA RNA

source_name: IRF4-deficient B cells from division 6

extract_protocol:
Cells were FACS sorted into RLT + 1%BME and snap frozen using the indicated markers. Total RNA was purified using the Quick-RNA MicroPrep Kit (Zymo Research)
All purified RNA was used as input for the SMART-seq v4 cDNA synthesis kit (Takara). Final libraries were constructed using 400 pg of cDNA as input for the NexteraXT kit with the NexteraXT indexing primers (Illumina).

treatment_protocol: Splenic naive IRF4-sufficent and -deficient B cells were isolated (Miltenyi CD43- isolation kit), CTV-labeled, and 15 million cells were transferred into mMT hosts. One day later host mice were challenged with LPS, and three days post-LPS challenge cells were isolated in discrete division by the indicated markers.

tag: Sex

value: Female

tag: sorting markers

tag: genotype/variation

value: Cd19cre/+ Irf4fl/fl

tag: tissues for b cell isolation and recovered from hosts

value: Spleen

tag: host

value: uMT

tag: stimulation

value: LPS

tag: days post-lps inoculation of host


title: stool small RNA-Seq VOV134

tag: diet


title: Free-MNase in WT, rep-3

tag: chec tag

tag: strain


title: Free-MNase in WT, rep-2


title: stool small RNA-Seq VOV131


title: Free-MNase in WT, rep-1


title: m6_div5: Bcell-Div-CrePos_4_d5_RNAseq

source_name: IRF4-deficient B cells from division 5


title: stool small RNA-Seq VOV130


title: Free-MNase in  med16-deletion, rep-3


title: Free-MNase in  med16-deletion, rep-2


title: MED13 in WT, rep-3.1


title: m6_div4: Bcell-Div-CrePos_4_d4_RNAseq

source_name: IRF4-deficient B cells from division 4


title: Free-MNase in  med16-deletion, rep-1


title: stool small RNA-Seq VOV12


title: MED13 in WT, rep-2.1


title: Wi38VIS3

description:
VIS Wi38
Wi38VIS3
VIS_ctrl_Wi38_norm_counts

tag: condition

tag: infection

value: direct virus infection


title: Wi38VIS2

description:
VIS Wi38
Wi38VIS2
VIS_ctrl_Wi38_norm_counts


title: m6_div3: Bcell-Div-CrePos_4_d3_RNAseq

source_name: IRF4-deficient B cells from division 3


title: Wi38VIS1

description:
VIS Wi38
Wi38VIS1
VIS_ctrl_Wi38_norm_counts


title: stool small RNA-Seq VOV132


title: m6_div0: Bcell-Div-CrePos_4_d0_RNAseq

source_name: IRF4-deficient B cells from division 0


title: Wi38ctrl2

description:
control Wi38
Wi38ctrl2
VIS_ctrl_Wi38_norm_counts


title: Wi38ctrl3

description:
control Wi38
Wi38ctrl3
VIS_ctrl_Wi38_norm_counts


title: stool small RNA-Seq VOV127


title: stool small RNA-Seq VOV129


title: stool small RNA-Seq VOV126


title: 9-RNAseq-577

description:
cells cult. with CM of VIS IMR90 fibs, CD86 depleted fraction/flow-through of MACS beads
CM_IMR90_VIS_CD86_minus1
THP1_norm_counts

value: direct SARS-CoV-2 infection


title: Wi38ctrl1

description:
control Wi38
Wi38ctrl1
VIS_ctrl_Wi38_norm_counts


title: m5_div6: Bcell-Div-CrePos_2_d6_RNAseq


title: stool small RNA-Seq VOV125


title: 8-RNAseq-576

description:
cells cult. with CM of VIS Wi38 fibs, MACS-isolated CD86+ fraction
CM_Wi38_VIS_CD86_plus2
THP1_norm_counts


title: m5_div5: Bcell-Div-CrePos_2_d5_RNAseq


title: 8-RNAseq-573

description:
cells cult. with CM of VIS Wi38 fibs, MACS-isolated CD86+ fraction
CM_Wi38_VIS_CD86_plus1
THP1_norm_counts


title: 7-RNAseq-571

description:
cells cult. with CM of VIS Wi38 fibs, CD86 depleted fraction/flow-through of MACS beads
CM_Wi38_VIS_CD86_minus1
THP1_norm_counts


title: stool small RNA-Seq VOV122


title: 7-RNAseq-572

description:
cells cult. with CM of VIS Wi38 fibs, CD86 depleted fraction/flow-through of MACS beads
CM_Wi38_VIS_CD86_minus2
THP1_norm_counts


title: m6_div1: Bcell-Div-CrePos_4_d1_RNAseq

source_name: IRF4-deficient B cells from division 1


title: m5_div3: Bcell-Div-CrePos_2_d3_RNAseq


title: stool small RNA-Seq VOV124


title: m5_div4: Bcell-Div-CrePos_2_d4_RNAseq


title: 4-RNAseq-569

description:
supernatant/conditioned medium of proliferating Wi38 fibroblasts
SN_Wi381
THP1_norm_counts


title: m5_div1: Bcell-Div-CrePos_2_d1_RNAseq


title: 2-RNAseq-568

description:
supernatant/conditioned medium of proliferating IMR90 fibroblasts
SN_IMR902
THP1_norm_counts


title: 2-RNAseq-567

description:
supernatant/conditioned medium of proliferating IMR90 fibroblasts
SN_IMR901
THP1_norm_counts


title: m4_div6: Bcell-Div-CrePos_1_d6_RNAseq


title: 10-RNAseq-503

description:
cells cult. with CM of VIS IMR90 fibs, MACS-isolated CD86+ fraction
CM_IMR90_VIS_CD86_plus2
THP1_norm_counts


title: 10-RNAseq-502

description:
cells cult. with CM of VIS IMR90 fibs, MACS-isolated CD86+ fraction
CM_IMR90_VIS_CD86_plus1
THP1_norm_counts


title: 9-RNAseq-501

description:
cells cult. with CM of VIS IMR90 fibs, CD86 depleted fraction/flow-through of MACS beads
CM_IMR90_VIS_CD86_minus2
THP1_norm_counts


title: stool small RNA-Seq VOV121


title: stool small RNA-Seq VOV119

value: Omnivorous


title: stool small RNA-Seq VOV19


title: m4_div5: Bcell-Div-CrePos_1_d5_RNAseq


title: 1-RNAseq-566

description:
control THP1
control2
THP1_norm_counts


title: stool small RNA-Seq VOV117


title: YP_9d_rep2

tag: growth medium

value: Yeast extract - peptone (YP)

tag: incubation time

value: 9 days

tag: sequencing platform

value: HiSeq


title: m4_div4: Bcell-Div-CrePos_1_d4_RNAseq


title: YP_9d_rep1


title: 4-RNAseq-570

description:
supernatant/conditioned medium of proliferating Wi38 fibroblasts
SN_Wi382
THP1_norm_counts


title: stool small RNA-Seq VOV116


title: YP_5d_rep2

value: 5 days


title: m4_div3: Bcell-Div-CrePos_1_d3_RNAseq


title: YP_2d_rep2

value: 2 days


title: YP_5d_rep1


title: stool small RNA-Seq VOV11


title: stool small RNA-Seq VOV115


title: m4_div1: Bcell-Div-CrePos_1_d1_RNAseq


title: YP_2d_rep1


title: stool small RNA-Seq VOV114

value: Vegan


title: stool small RNA-Seq VOV113


title: 1-RNAseq-565

description:
control THP1
control1
THP1_norm_counts


title: m3_div5: Bcell-Div-CreNeg_3_d5_RNAseq

source_name: IRF4-sufficient B cells from division 5

value: Cd19+/+ Irf4fl/fl


title: m3_div6: Bcell-Div-CreNeg_3_d6_RNAseq

source_name: IRF4-sufficient B cells from division 6


title: stool small RNA-Seq VOV111

value: Vegetarian


title: GRN/TREM2 KO 78

city: Munich

first: Anika

last: Reifschneider

address: Feodor-Lynen-Straße

institute: LMU

description: nCounter® Neuropathology panel

data_processing: NanoString data were normalized and analyzed using nSolver™ software version 4.0 (NanoString Technologies, Inc.).  The geometric mean of the 10 on-chip housekeeping genes was used for normalization of reads.

source_name: total brain

growth_protocol: Housing conditions included standard pellet food and water provided ad libitum, 12-hour light-dark cycle at temperature of 22 °C with maximal 5 mice per cage and cage replacement once per week and regular health monitoring.

extract_protocol: Total RNA was isolated using Qiagen RNeasy Mini Kit according to the manufacturer's protocol

value: female

value: Grn/Trem2 double knockout


title: GRN/TREM2 KO 37

description: nCounter® Neuropathology panel


title: GRN/TREM2 KO 36

description: nCounter® Neuropathology panel


title: m3_div4: Bcell-Div-CreNeg_3_d4_RNAseq

source_name: IRF4-sufficient B cells from division 4

value: Female


title: TREM2 KO 447

description: nCounter® Neuropathology panel

value: Trem2 knockout


title: TREM2 KO 363

description: nCounter® Neuropathology panel


title: GRN/TREM2 KO 14

description: nCounter® Neuropathology panel


title: m3_div3: Bcell-Div-CreNeg_3_d3_RNAseq

source_name: IRF4-sufficient B cells from division 3

value: Spleen

value: uMT


title: TREM2 KO 601

description: nCounter® Neuropathology panel


title: TREM2 KO 357

description: nCounter® Neuropathology panel


title: GRN KO 17

description: nCounter® Neuropathology panel

value: Grn knockout


title: stool small RNA-Seq VOV110

source_name: Stool


title: m4_div0: Bcell-Div-CrePos_1_d0_RNAseq

value: Cd19cre/+ Irf4fl/fl

value: LPS


title: m3_div1: Bcell-Div-CreNeg_3_d1_RNAseq

source_name: IRF4-sufficient B cells from division 1


title: stool small RNA-Seq VOV112


title: stool small RNA-Seq VOV109

value: Omnivorous


title: GRN KO 54

description: nCounter® Neuropathology panel


title: m3_div0: Bcell-Div-CreNeg_3_d0_RNAseq

source_name: IRF4-sufficient B cells from division 0


title: stool small RNA-Seq VOV108


title: GRN KO 560

description: nCounter® Neuropathology panel


title: WT 650

description: nCounter® Neuropathology panel

value: WT


title: GRN KO 559

description: nCounter® Neuropathology panel


title: WT 537

description: nCounter® Neuropathology panel


title: m2_div6: Bcell-Div-CreNeg_2_d6_RNAseq


title: stool small RNA-Seq VOV107


title: m2_div5: Bcell-Div-CreNeg_2_d5_RNAseq


title: m2_div4: Bcell-Div-CreNeg_2_d4_RNAseq


title: WT 536

description: nCounter® Neuropathology panel


title: stool small RNA-Seq VOV105


title: m2_div3: Bcell-Div-CreNeg_2_d3_RNAseq


title: WT 15

description: nCounter® Neuropathology panel


title: stool small RNA-Seq VOV104


title: stool small RNA-Seq VOV10


title: m2_div1: Bcell-Div-CreNeg_2_d1_RNAseq


title: stool small RNA-Seq VOV102


title: stool small RNA-Seq VOV142


title: stool small RNA-Seq VOV101


title: m2_div0: Bcell-Div-CreNeg_2_d0_RNAseq


title: m1_div6: Bcell-Div-CreNeg_1_d6_RNAseq


title: stool small RNA-Seq VOV106


title: m1_div5: Bcell-Div-CreNeg_1_d5_RNAseq


title: stool small RNA-Seq VOV92


title: stool small RNA-Seq VOV100


title: m1_div4: Bcell-Div-CreNeg_1_d4_RNAseq


title: ATF-silenced cells, biological rep 2

city: Durham

first: Jen-Tsan

middle: Ashley

last: Chi

state: NC

address: 101 Science Drive, DUMC 3382

institute: Duke University

description:
Human RCC4 cells with ATF4 silenced by siRNA
siATF4-2
3720_17223_46892_3-2

hyb_protocol: cDNA was amplified using Ambion MessageAmp™ Premier RNA Amplification Kit (Life Technologies) from 200ng total RNA, hybridized to GeneChip U133A 2.0 arrays in Affymetrix  GeneChip® hybridization oven 645, washed in Affymetrix GeneChip®  Fluidics Station 450

scan_protocol: GeneChips were scanned with Affymetrix GeneChip® Scanner 7G.

data_processing: The data were analyzed with Expression Console and normalized by the RMA (Robust Multi-Array) algorithm. The influence of the silencing of MESH1 and ATF4 on gene expression was derived by a zero transformation process, in which we compared the gene expression level with siMESH1 or siMESH1+siATF4 to the average of gene expression level in control (siNT) samples.

label: biotin

source_name: ATF-silenced cells

label_protocol: Standard Affymetrix protocol

growth_protocol: RCC4 cells were grow in DMEM media with 4.5g/L glucose and 4mM glutamine (11995-DMEM, ThermoFisher Scientific) and supplement with 10% heat-inactivated fetal bovine serum (Hyclone #SH30070.03HI) in humidified incubator, 37°C with 5% CO2

extract_protocol: Total RNA was extracted using RNeasy Mini Kit (Qiagen, #74104) according to the manufacturer's instructions.

treatment_protocol: Human clear cell renal carcinoma cell line RCC4 were transfected with siRNA targeting MESH1 (D-031786-01,  Dharmacon), ATF4 (M-005125-02, Dharmacon), or MESH1 with ATF4 together, and compared with non-targeting siRNA control (AllStars Negative Control siRNA, SI03650318). Briefly, 10^5 RCC4 cells were seeded in a well on 6-well plate with 40 pmole of siRNA and 3μL of Lipofectamine RNAiMAX (ThermoFisher Scientific, #133778150) reagent. Cells were incubated for 72 hours before collection.

tag: cell line

value: clear cell renal caricnoma cells

tag: genotype

value: low ATF4


title: MESH1-silenced cells, biological rep 2

description:
Human RCC4 cells with MESH1 silenced by siRNA
siMESH1-2
3720_17223_46889_2-2

source_name: MESH1-silenced cells

tag: cell type

value: low MESH1


title: ATF-silenced cells, biological rep 1

description:
Human RCC4 cells with ATF4 silenced by siRNA
siATF4-1
3720_17223_46891_3-1


title: MESH1-silenced cells, biological rep 3

description:
Human RCC4 cells with MESH1 silenced by siRNA
siMESH1-3
3720_17223_46890_2-3


title: ATF-silenced cells, biological rep 3

description:
Human RCC4 cells with ATF4 silenced by siRNA
siATF4-3
3720_17223_46893_3-3


title: MESH1-silenced cells, biological rep 1

description:
Human RCC4 cells with MESH1 silenced by siRNA
siMESH1-1
3720_17223_46888_2-1


title: stool small RNA-Seq VOV18

value: Vegan


title: stool small RNA-Seq VOV91


title: MESH1 and ATF4 silenced cells, biological rep 1

description:
Human RCC4 cells with MESH1 and ATF4 silenced by siRNA
siMESH1+siATF4-1
3720_17223_46894_4-1

source_name: MESH1 and ATF4 silenced cells

value: low MESH1 and ATF4


title: m1_div3: Bcell-Div-CreNeg_1_d3_RNAseq

tag: Sex

tag: sorting markers

value: Cd19+/+ Irf4fl/fl


title: non-targeting siRNA control, biological rep 3

description:
Human RCC4 cells with non-targeting siRNA control
siNT-3
3720_17223_46887_1-3

source_name: non-targeting siRNA control

value: control


title: MESH1 and ATF4 silenced cells, biological rep 3

description:
Human RCC4 cells with MESH1 and ATF4 silenced by siRNA
siMESH1+siATF4-3
3720_17223_46896_4-3


title: stool small RNA-Seq VOV81


title: MESH1 and ATF4 silenced cells, biological rep 2

description:
Human RCC4 cells with MESH1 and ATF4 silenced by siRNA
siMESH1+siATF4-2
3720_17223_46895_4-2


title: non-targeting siRNA control, biological rep 1

description:
Human RCC4 cells with non-targeting siRNA control
siNT-1
3720_17223_46885_1-1


title: non-targeting siRNA control, biological rep 2

description:
Human RCC4 cells with non-targeting siRNA control
siNT-2
3720_17223_46886_1-2


title: VC214

hyb_protocol: 1 µg of purified cRNA from samples and reference were hybridized on the oligo glass two-color array using Agilent Technologies SurePrint G3 Human Gene Expression Microarrays 4X44K v2. The dye-swap replication procedure was applied.

scan_protocol: Images were scanned using an Agilent C dual-laser microarray scanner and analyzed by the Agilent Feature Extraction (FE) Software (Agilent Technologies).

data_processing: The LIMMA (LInear Models for Microarray Analysis) package was used to analyze gene expression profile of oropharynx squamous cell carcinoma samples. Raw intensity values were background subtracted (method = normexp, offset = 50) and normalized using the loess method, for the within array normalizaton, and Aquantile, for the between array normalization. Duplicated probes were averaged. Then, Cy3 and Cy5 channels were separated using the separate channel analysis of two-color data, available within the Limma package.

label_protocol: Five hundred ng of total RNA of each sample and of Universal Reference (Human Universal Reference Total RNA; Clontech, Mountain View, CA) were amplified by the Amino Allyl MessageAmp I aRNA Kit (Ambion, Austin, TX), as previously described [20]. Labeling was performed using cyanine 3 (IRIS 3) and cyanine 5 (IRIS 5) (Molecular Targeting Technologies, Inc).

extract_protocol: Briefly, fresh frozen tissues were fragmented through mechanical homogenization by using TissueLyser LT (Qiagen) and addition of TRI Reagent (Sigma Aldrich). Total RNA extraction and isolation was performed by using the Absolutely RNA miRNA kit (Agilent Technologies) following the manufacturer’s instructions. The quality of total RNA was assessed by capillary electrophoresis with Bioanalyzer 2100 (Agilent Technologies); Purity and quantity of total RNA were assessed using NanoDrop spectrophotometer (NanoDrop Thermo Scientific).

tag: tissue

value: Oropharynx squamous cell carcinoma

tag: hpv status

value: Negative

tag: survival

value: deceased

source_name: Ref

value: Universal Reference


title: m1_div1: Bcell-Div-CreNeg_1_d1_RNAseq

value: Female

tag: genotype/variation

tag: tissues for b cell isolation and recovered from hosts

value: Spleen

tag: host

value: uMT

tag: stimulation

tag: days post-lps inoculation of host


title: stool small RNA-Seq VOV79

tag: diet

value: Vegetarian


title: m1_div0: Bcell-Div-CreNeg_1_d0_RNAseq

value: LPS


title: VC214 Dye Swap


title: stool small RNA-Seq VOV77

value: Omnivorous


title: VC213 Dye Swap

value: Positive


title: stool small RNA-Seq VOV62


title: m7_div6_ATACseq

data_processing:
Raw sequencing reads were mapped to the mm10 version of the mouse genome using Bowtie-1.1.1
For each sample peaks of accessibility were identified and annotated using MACS2 with the default settings
Genome_build: mm10
Supplementary_files_format_and_content: bigwig files are normalized to reads per peak million (rpm) using R/Bioconductor

source_name: IRF4-deficient B cells from division 6

extract_protocol:
Cells were FACS sorted into FACS buffer and tagmentation for ATAC-seq performed.
10,000 cells were centrifuged for 10 minutes at 500xg, and tagmentation was performed by resuspending cells in 25 ul of Tagmentation mix (1X TD buffer (10 mM TAPS-NaOH pH 8.1, 5 mM MgCl2, 10% DMF), 2.5 μl Tn5 transposase, 0.1% Tween 20 and 0.02% Digitonin) and then incubated for 1 h at 37°C. DNA was purified and amplified into a sequencing library with KAPA HiFi Polymerase and the Nextera Indexing Kit Primers (Illumina, Inc).

value: Cd19cre/+ Irf4fl/fl


title: stool small RNA-Seq VOV39


title: stool small RNA-Seq VOV13


title: VC213


title: VC211 Dye Swap

value: Non Evidence of Disease


title: m7_div4_ATACseq

source_name: IRF4-deficient B cells from division 4


title: VC212 Dye Swap


title: VC211


title: VC212


title: m7_div3_ATACseq

source_name: IRF4-deficient B cells from division 3


title: stool small RNA-Seq VOV4


title: VC209 Dye Swap


title: m7_div1_ATACseq

source_name: IRF4-deficient B cells from division 1


title: m7_div5_ATACseq

source_name: IRF4-deficient B cells from division 5


title: VC210-II Dye Swap


title: VC208 Dye Swap


title: m7_div0_ATACseq

source_name: IRF4-deficient B cells from division 0


title: VC209


title: VC208


title: VC207 Dye Swap


title: VC206 Dye Swap


title: VC207


title: SUDHL4_CD40_IgM_rep2

city: New York

first: David

last: Dominguez-Sola

state: NY

address: One Gustave L. Levy Place, Box 1044A

department: Oncological Sciences

institute: Icahn School of Medicine at Mount Sinai

description: SUDHL4 Cells stimulated with megaCD40L and IgM/G for 24 hours

data_processing:
Sequencing data were acquired through the default Illumina pipeline using bcl2fastq pipeline
reads were aligned to human genome reference hg19 using TopHat with options –read-mismatches 2 –read-edit-dist 2 –max-multihits 10
Htseq-count was used to generate gene counts
Genome_build: grch37
Supplementary_files_format_and_content: Tab delimited text file of raw gene counts

growth_protocol: Cells were maintained in log-phase growth in a 37C incubator with 5% CO2 in growth media (IMDM, 10% FBS, 1% Penicillin/Strepromycin)

extract_protocol:
Total RNA was isolated using the PureLink RNA Extraction kit (Qiagen), according to the manufacturer's instructions.
Libraries were prepared by Novogene

treatment_protocol: Treatment with combined megaCD40L (100ng/mL) and IgM/G (3.33 ug/mL ) for 24 hours

value: DLBCL cell

tag: protocol

value: megaCD40L and IgM/G


title: VC210-II


title: SUDHL4_control_rep2

description: SUDHL4 Cells grown in normal media (IMDM+10%FBS+pen/strep)

value: normal media


title: SUDHL4_CD40_IgM_rep1


title: m6_div5_ATACseq


title: SUDHL4_control_rep1


title: VC205 Dye Swap


title: VC205


title: VC204 Dye Swap


title: m6_div3_ATACseq


title: m6_div6_ATACseq


title: VC206


title: VC204


title: m6_div0_ATACseq


title: S1 refers to 3ae-treated group in manuscript

city: Hong Kong

first: Zihao

last: Wang

address: 224 Waterloo Road

department: School of Chinese Medicine

country: Hong Kong

institute: Hong Kong Baptist University

data_processing:
Then libraries with different indices were multiplexed and loaded on an Illumina HiSeq instrument according to manufacturer’s instructions (Illumina, San Diego, CA, USA).
Sequencing was carried out using a 2x150bp paired-end (PE) configuration; image analysis and base calling were conducted by the HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) on the HiSeq instrument.
The sequences were processed and analyzed by GENEWIZ.
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

extract_protocol:
RAW 264.7 cells were treated with each compound (25, 50 mM) for 24 h followed by LPS (1 mg/mL) stimulation for another 2 h. Cells were collected and then directly prepared for cDNAs amplification and RNA-Seq library construction. Total RNA of each sample was extracted using TRIzol Reagent (Invitrogen)/RNeasy Mini Kit (Qiagen)/other kits. Total RNA of each sample was quantified and qualified by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), NanoDrop (Thermo Fisher Scientific Inc.) and 1% agrose gel. 1 μg total RNA with RIN value above 6.5 was used for following library preparation.
Next generation sequencing library preparations were constructed according to the manufacturer’s protocol.

treatment_protocol: Complete chemical names and structures for treatments are included in the file GEO_chemical name_WZH.docx, available on the series record.

tag: treatment


title: m6_div4_ATACseq


title: R1 refers to 3m-treated group in manuscript


title: Q1 refers to 3o-treated group in manuscript


title: P1 refers to 3ag-treated group in manuscript


title: m5_div6_ATACseq


title: U1 refers to 3p-treated group in manuscript


title: VC202

value: relapse


title: VC203 Dye Swap

value: Oropharynx squamous cell carcinoma


title: VC202 Dye Swap

value: Negative


title: VC200 Dye Swap


title: VC200


title: VC201


title: VC201 Dye Swap


title: NO313 Dye Swap


title: m6_div1_ATACseq


title: M1 refers to LPS-stimulated group in manuscript


title: NO312


title: NO312 Dye Swap


title: NO310

value: deceased


title: NO310 Dye Swap

value: Universal Reference


title: NO313


title: m5_div3_ATACseq

value: Female


title: Lymphoid_Control

city: Goyang-si

first: Chungyong

last: Han

state: Gyeonggi-do

address: 323 Ilsan-ro, Ilsandong-gu

country: South Korea

institute: National Cancer Center

description: mixed sample (non-demultiplexed)

data_processing:
The Illumina sequencer generates raw images utilizing sequencing control software for system control and base calling through an integrated primary analysis software called RTA (Real Time Analysis). The BCL (base calls) binary is converted into FASTQ utilizing illumina package bcl2fastq. Adapters are not trimmed away from the reads.
Data processing was performed by running the BD Rhapsody™ Analysis pipelines for sequencing on the Seven Bridges Genomics platform. See details in the BD Single-Cell Multiomics Bioinformatics Handbook (Doc ID: 54169).
In brief, read pairs with low sequencing quality are first removed: If the length of R1 is >75 bases, the read is trimmed to 75 bases; If the length of R1 read is <60 bp or R2 read is <42 bp, the R1/R2 read pair is dropped; If the mean base quality score of either R1 read or R2 read is <20, the read pair is dropped; If single nucleotide frequency is ≥0.55 for the R1 read or SNF ≥0.80 for the R2 read, the read pair is dropped.
The quality-filtered R1 reads were analyzed to identify the cell label section sequence (CLS), common sequences (L), Unique Molecular Identifier (UMI) sequence, and if applicable, poly(T) tail. STAR (Spliced Transcripts Alignment to a Reference) was used to map the filtered R2 reads to the transcriptome.
Reads with the same cell label, same UMI sequence, and same gene are collapsed into a single raw molecule. To distinguish cell labels associated with putative cells from those associated with noise, a multi-step algorithm was used to filter cell labels. Fox multiplexing samples, the analysis pipeline automatically added the Sample Tag sequences to the FASTA reference file. Reads that aligned to a Sample Tag sequence and associated with a putative cell were used to identify the sample for that cell. Molecule counts and associated reads of each gene for each putative cell were recorded in .csv format.
Genome_build: mm10
Supplementary_files_format_and_content: CSV format (Combined_CTXCD4_S1_LaneData_RSEC_MolsPerCell). Each row represents the number of molecules in a cell for each gene detected. A cell is identified with a unique cell index number under Cell_Index.
Supplementary_files_format_and_content: CSV format (CTXCD4_S1_LaneData_Putative_Cells_Origin). This output lists the step in the cell label filtering algorithm that determined a particular cell is a putative cell. If the cell label is categorized as putative in the basic implementation of the second derivative analysis, it is labeled Basic. If the cell label is a recovered false negative in the refined implementation, it is labeled Refined.
Supplementary_files_format_and_content: CSV format (CTXCD4_S1_LaneData_Sample_Tag_Calls). This file contains the determined sample call (multiplexing) for every putative cell.

source_name: Lymphoid cells

growth_protocol: On day 0, six- to eight-week-old C57BL/6 female mice were intradermally inoculated with B16F10 (2E+05). On day 3, pre-conditioning lymphodepletion was induced via intraperitoneal injection of 300 mg/kg cyclophosphamide. On day 5, Pmel-1 cells (2E+06) were isolated from the splenocytes of Thy1.1 Pmel-1 transgenic mice (Thy1a/Cy Tg(TcraTcrb)8Rest/J; expressing transgenic TCR specific for gp10025–33 and congenic Thy1.1 antigen) and injected into tumor-bearing mice. Starting on day 5, recombinant human IL-2 (10,000 IU) was intraperitoneally injected daily for 3 days.

extract_protocol:
On day 25, the lymph nodes and spleen were gently disrupted and filtered through a 40 μm nylon cell strainer to make single-cell suspensions. Cells were treated with RBC lysis buffer before use. For each group (control and aCD4), whole lymphoid cells, Thy1.1-negative endogenous CD8+ T cells, and Thy1.1-positive Pmel-1 cells were subjected to the analysis. Endogenous CD8+ T cells and Pmel-1 cells were isolated using anti-Thy1.1 and CD8 isolation microbeads. A total of six samples (3 populations for each group) were multiplexed using a BD mouse immune single-cell multiplexing kit. Poly-A RNA of each sample was extracted using lysis buffer in a BD Rhapsody cDNA kit.
cDNA library was constructed using the BD Rhapsody system, a BD Rhapsody WTA amplification kit, and a BD Rhapsody cDNA kit according to the manufacturer's protocol.

treatment_protocol: Starting from day 10, anti-CD4 treatment group involved weekly intraperitoneal injections of 200 μg anti-mouse CD4 monoclonal antibody (clone: GK1.5) for 3 weeks.

tag: strain

tag: gender

tag: age

value: Six- to eight-week-old

value: Inguinal lymph nodes and spleen

value: Lymphoid_Control, Lymphoid_aCD4, Endogenous_Control, Endogenous_aCD4, Pmel1_Control, Pmel1_aCD4


title: m5_div1_ATACseq

value: Spleen

value: uMT

value: LPS


title: NO306

value: Positive


title: m5_div5_ATACseq

value: Cd19cre/+ Irf4fl/fl


title: 3_Neg

city: Rochester

first: Cameron

last: Baker

address: 601 Elmwood Ave

institute: University of Rochester

data_processing:
062918 BATCH - Trimmomatic-0.36, TRAILING:13 LEADING:13 ILLUMINACLIP:adapters.fasta:2:30:10 SLIDINGWINDOW:4:20 MINLEN:35
062918 BATCH - STAR_2.6.0c with the following parameters: "—twopass Mode Basic --runMode alignReads --outSAMtype BAM SortedByCoordinate – outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonical –outReads UnmappedFastx"
062918 BATCH - subread-1.6.1 package (featureCounts) with the following parameters: "-s 0 -t exon -g gene_name"
062918 BATCH - Differential expression analysis was performed using DESeq2-1.22.1
020520 BATCH - FastP version 0.20.0 with the following parameters: "--length_required 35 --cut_front_window_size 1 --cut_front_mean_quality 13 --cut_front --cut_tail_window_size 1 --cut_tail_mean_quality 13 --cut_tail -y -r"
020520 BATCH - STAR_2.7.0f with the following parameters: "—twopass Mode Basic --runMode alignReads --outSAMtype BAM SortedByCoordinate – outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonical –outReads UnmappedFastx"
020520 BATCH - subread-1.6.4 package (featureCounts) with the following parameters: "-s 0 -t exon -g gene_name"
020520 BATCH - Differential expression analysis was performed using DESeq2-1.22.1
Genome_build: 062918 BATCH - GRCm38.p6 + Gencode-M217Annotation, 020520 BATCH - GRCm38.p6 + Gencode-M22 Annotation
Supplementary_files_format_and_content: Raw gene counts

source_name: Lung CD8 T cells

extract_protocol:
Intravenous labelling was conducted with anti-CD45 antibody before lung harvesting. After harvest and a single cell suspension was obtained, cells were either restimulated with anti-CD2/CD28 or not restimulated, then sorted into four groups based off of integrin expression. All cells were live, singlet, CD44+CD8+ cells.
Total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen, Valencia, CA) per manufacturer’s recommendations. RNA concentration was determined with the NanopDrop 1000 spectrophotometer (NanoDrop, Wilmington, DE) and RNA quality assessed with the Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA). 1ng of total RNA was pre-amplified with the SMARTer Ultra Low Input kit v4 (Clontech, Mountain View, CA) per manufacturer’s recommendations. The quantity and quality of the subsequent cDNA was determined using the Qubit Flourometer (Life Technnologies, Carlsbad, CA) and the Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA). 150pg of cDNA was used to generate Illumina compatible sequencing libraries with the NexteraXT library preparation kit (Illumina, San Diego, CA) per manufacturer’s protocols. The amplified libraries were hybridized to the Illumina single end flow cell and amplified using the cBot (Illumina, San Diego, CA). Single end reads of 100nt were generated by the HiSeq2500 (Illumina, San Diego, CA).

tag: cell type

value: Restimulated CD49a-CD103- CD8 T cell

value: Lung (intravenously labelled cells were excluded from collection)

tag: batch


title: 3_DP

value: Restimulated CD49a+CD103+ CD8 T cell


title: NO306 Dye Swap

value: Non Evidence of Disease


title: m5_div0_ATACseq

tag: Sex

tag: sorting markers


title: NO304

tag: tissue

value: Oropharynx squamous cell carcinoma

tag: hpv status


title: NO307

tag: survival


title: 3_CD49a

value: Restimulated CD49a+CD103- CD8 T cell


title: NO305 Dye Swap


title: m4_div4_ATACseq

tag: genotype/variation

tag: tissues for b cell isolation and recovered from hosts


title: NO307 Dye Swap


title: m4_div6_ATACseq

tag: host

tag: stimulation


title: NO305


title: NO304 Dye Swap

value: deceased

value: Negative


title: C1 refers to control group in manuscript

tag: cell line

value: control


title: 3_CD103

value: Restimulated CD49a-CD103+ CD8 T cell


title: NO303 Dye Swap

value: Universal Reference


title: NO303


title: m4_div3_ATACseq

value: Female

tag: days post-lps inoculation of host


title: 2_Neg


title: NO302 Dye Swap


title: m4_div1_ATACseq

value: Spleen


title: m5_div4_ATACseq

value: uMT


title: NO301-II Dye Swap


title: NO301-II


title: NO302


title: 2_DP


title: m4_div0_ATACseq

value: LPS


title: m3_div6_ATACseq

source_name: IRF4-sufficient B cells from division 6

value: Cd19+/+ Irf4fl/fl


title: NO300 Dye Swap


title: 2_CD49a


title: WT young microglia 2

city: Bonn

first: Melania

last: Capasso

address: Venusberg-Campus 1, Gebaeude 99

institute: DZNE e.V.

data_processing:
Illumina Casava for base calling and demultiplexing
Sequencing reads were adapter trimmed using Cutadapt 1.13 and aligned against mm10 (GENCODE release M14) using STAR aligner 2.5.3 with standard settings. For repeat element analysis STAR aligner was run with the following options: --outFilterMultimapNmax 100 --winAnchorMultimapNmax 100.
Reads mapping to rRNA, the mitochondrial genome and non-uniquely were removed. For repeat element expression analysis only reads mapping to the mitochondrial genome were removed.
Differentially expressed genes and repeat elements were obtained by running DESeq2 1.16.1.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited file with DESeq2 normalized counts for each sample

source_name: microglia

extract_protocol:
microglia were flow cytometry sorted from three separate mice per replicate, pooled prior to RNA extraction, which was carried out using an RNAeasy microkit (Qiagen), according to manufacturers’ guidelines.
Samples were prepared for RNA sequencing using the SMARTer Ultra Low RNA kit for Illumina sequencing (Clontech Laboratories) according to the manufacturer's instructions.

value: brain

tag: genotype

value: wild-type

value: 6 months

value: female


title: 2_CD103

value: Lung (intravenously labelled cells were excluded from collection)


title: BI103

value: Oropharynx squamous cell carcinoma

value: relapse


title: 1_Neg

value: Restimulated CD49a-CD103- CD8 T cell


title: WT young microglia 3

tag: strain


title: WT young microglia 1

tag: gender


title: WT old microglia 2

tag: age

value: 23 months


title: 1_DP

tag: cell type

value: Restimulated CD49a+CD103+ CD8 T cell


title: WT old microglia 3


title: m3_div4_ATACseq

source_name: IRF4-sufficient B cells from division 4


title: m3_div3_ATACseq

source_name: IRF4-sufficient B cells from division 3


title: WT old microglia 1


title: WT middle aged microglia 3

value: 15 months


title: m3_div1_ATACseq

source_name: IRF4-sufficient B cells from division 1


title: WT middle aged microglia 2


title: NO300

value: Negative

value: deceased

value: Universal Reference


title: BI103 Dye Swap


title: m3_div0_ATACseq

source_name: IRF4-sufficient B cells from division 0

value: Female

value: Spleen

value: uMT


title: m3_div5_ATACseq

source_name: IRF4-sufficient B cells from division 5

value: Cd19+/+ Irf4fl/fl

value: LPS


title: 34194: keratinocytes ko-3

city: Vienna

first: Christian

last: Seiser

address: Schwarzspanierstrasse 17

department: Center for Anatomy and Cell Biology

institute: Medical University of Vienna

data_processing:
SCS [PN:HiSeq Control Software;;VN:2.2.58;;Controlling software on instrument]
basecalling [PN:RTA;;VN:1.18.64.0;;Basecalling Package]
bcl2fastq [PN:bcl2fastq;;VN:2.16.0;;bcl2fastq;;--runfolder-dir /projects/hiseq/hiseq5/Runs/FC_B/151231_D00689_0119_BC8918ANXX --output-dir /projects/hiseq/hiseq5/Runs/FC_B/151231_D00689_0119_BC8918ANXX/Data/Intensities/BaseCalls/illumina2bam0.03_02-01-2016 --tiles s_1 --loading-threads 4  --processing-threads 4  --writing-threads 1  --demultiplexing-threads 1]
fastq2bam [PN:fastq2bam;;VN:0.3;;conversion to unaligned bam files;;--runfolder /projects/hiseq/hiseq5/Runs/FC_B/151231_D00689_0119_BC8918ANXX --outputDir /projects/hiseq/hiseq5/Runs/FC_B/151231_D00689_0119_BC8918ANXX/Data/Intensities/BaseCalls/illumina2bam0.03_02-01-2016 --lane 1 --date 02-01-2016 --libraries M2885,34189=ACAGTG,34190=TGACCA --samples M2885,34189=ACAGTG,34190=TGACCA,M0,35026=CGATGT]
BamIndexDecoder [PN:BamIndexDecoder;;VN:V1.16-1-g590b59e-dirty;;demultiplexging;;uk.ac.sanger.npg.picard.BamIndexDecoder INPUT=/projects/hiseq/hiseq5/Runs/FC_B/151231_D00689_0119_BC8918ANXX/Data/Intensities/BaseCalls/illumina2bam0.03_02-01-2016/C8918ANXX_1_02-01-2016.bam OUTPUT_DIR=C8918ANXX_20151231B_demux_1 OUTPUT_PREFIX=C8918ANXX_1 OUTPUT_FORMAT=bam BARCODE_FILE=C8918ANXX_20151231B_demux_1/C8918ANXX_1_barcodes.tab METRICS_FILE=C8918ANXX_20151231B_demux_1/C8918ANXX_1_metrics.tab MAX_MISMATCHES=1 MIN_MISMATCH_DELTA=1 MAX_NO_CALLS=1 COMPRESSION_LEVEL=9 MAX_RECORDS_IN_RAM=60000000 CREATE_MD5_FILE=true    BARCODE_TAG_NAME=BC BARCODE_QUALITY_TAG_NAME=QT CONVERT_LOW_QUALITY_TO_NO_CALL=false MAX_LOW_QUALITY_TO_CONVERT=15 VERBOSITY=INFO QUIET=false VALIDATION_STRINGENCY=STRICT CREATE_INDEX=false]
bam2fastq [PN:samtools;;VN:1.3.1;;conversion to fastq]
cutadapt [PN:cutadapt;;VN:v1.4.2;;adapter trimming;;--match-read-wildcards -O 1 -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC]
bowtie2 [PN:bowtie2;;VN:2.1.0 ;;rRNA removal by alignment to rDNA;;--very-sensitive-local;;KY962518.1, NR_023363.1 and the ERCC.fa]
star [PN:STAR;;VN:v2.4.2a;;mRNA alignment;;--outSAMstrandField None --outFilterIntronMotifs RemoveNoncanonical --outFilterMismatchNoverLmax 0.1 --outFilterMismatchNmax 10 --outFilterScoreMinOverLread 0.30 --outFilterMatchNminOverLread 0.30 --outFilterMatchNmin 30 --chimSegmentMin 15 --quantMode TranscriptomeSAM --chimJunctionOverhangMin 15 --twopassMode Basic --outSAMtype SAM --outSAMattributes All --outReadsUnmapped Fastx intronMotif --alignIntronMax 200000  --outSJfilterIntronMaxVsReadN 10000 20000 30000 50000 --outSJfilterOverhangMin 20 12 12 12 --outFilterType BySJout --alignMatesGapMax 0  --outFilterMultimapNmax 20]
htseq [PN:htseq-count ;;VN:0.6.0;;read counting;;--mode union --stranded reverse]
combine [PN:combine.py;;VN:0.3.0;;combines counts to table]
repenrich [PN:RepEnrich;;VN:commit 0e9228149b2aff06874ed7ff0c8836aefa864c9e;;repeat enrichment]
diff [PN:deseq2;;VN:1.10.1;;differential expression]
transcriptome annotation: ENSEMBL Mus_musculus.GRCm38.79
Genome_build: GRCm38

source_name: keratinocytes

growth_protocol: For RNA sequencing epidermis of three control and three Dnmt1∆/∆ep mice was isolated at P3 and keratinocytes were cultured for 5 days. Mouse epidermal keratinocytes were isolated as previously described (Sibilia et al, 2000) and cultured in keratinocyte growth medium (KGM BulletKit©, Clonetics©) containing 8% chelated fetal calf serum and 0.05mM CaCl2 on collagen-fibronectin coated dishes. Total RNA was isolated following the manufacturer’s instructions (TRIzol, Invitrogen) and then used for RNA sequencing.

extract_protocol:
Total RNA was isolated following the manufacturer’s instructions (TRIzol, Invitrogen) and then used for RNA sequencing.
For transcriptome sequencing (RNA-seq) experiments, RNA was subjected to poly(A) selection with a Dynabeads mRNA purification kit (Invitrogen), followed by reverse transcription using a NEB RNA Ultra kit and library generation using a TruSeq library generation kit (Illumina).

treatment_protocol: keratinocytes were cultured for 5 days and then harvested for RNA isolation.

value: Dmnt1 knock-out

value: keratinocytes


title: 1_CD103

value: Restimulated CD49a-CD103+ CD8 T cell

value: Lung (intravenously labelled cells were excluded from collection)

tag: batch


title: WT middle aged microglia 1

value: brain

value: wild-type

value: female


title: tsRNA_1M_S8_RPI40

city: Bethesda

first: Pavol

last: Genzor

address: 6720A Rockledge Drive, Suite 100

department: ACESO

institute: Henry M Jackson Foundation

description:
total small RNA
size selected total small RNAs with 10 UMIs containing callibrator oligos
piRNA_quantification.xlsx

data_processing:
Fastq files were trimmed using cutadapt (v2.3) to remove the 5` and 3` adapters and keep 10 unique molecular identifiers (UMI) - 8N's at 5` end and 2N's at 3` end.
Fastq files were processed in R using custom script where sequences were collapsed by UMI to remove PCR duplicates, UMI's were trimmed,  and sequences shorter than 18-nt in length were removed. To reduce the file size while retaining all the information, the sequences were collapsed and individual sequence abundance was recorded in fasta header. Fasta header is comprised of sample name that is followed by a unique alphanumeric string containing sequence ID number (S) and abundance count (M) and have following structure: SAMPLE_XY - S[UniqueSequenceID]M[SequenceAbundance]. Fasta header "FH-Piwi-IP_1_1-S32M12" can be split into sample name ("FH-Piwi-IP_1_1") and unique string ("S32M12")  that can be further split into unique sequence ID ("S32") and abundance count of this particular sequence ("M12"). Please see UNIQSEQS.fa processed files. These files are deposited for the convenient, fast download and analysis.
UNIQSEQS.fasta files were aligned to genome comprised of the structural RNAs (tRNA, rRNA, snRNA, snoRNA) using STAR (v2.5.2b), the unmapped sequences were collected and mapped to the dm6 genome using the same aligner. The obtained .bam files were analyzed in R using custom scripts.
piRNA quantification was performed by determining the number of cells per sample (S1-S8) using molecular counts of the callibrator oligoes in the UNIQSEQS.fasta files. MiRNAs were quantified by mapping to miR Base (Release 22.1). PiRNAs were quantified by considering seqeunces in 24 - 29-nt size range.
Genome_build: dm6 (Drosophila melanogaster)
Supplementary_files_format_and_content: xlsx: piRNA_quantification; fasta: custom trimmed and filtered unique sequence files

organism: Drosophila melanogaster

source_name: OSC cell line

growth_protocol: OSC cell culture was grown as described in protocols described here: https://dgrc.bio.indiana.edu/product/View?product=288

extract_protocol:
piRNAs: RNA was extracted from the OSC cell lysate by immunoprecipitating PIWI proteins using anti-Flag antibody beads or custom made fly anti-Piwi antibodies. Total small RNAs: RNA was extracted from the cells usign PureLink miRNA Isolation kit (Thermo Fisher Scientific, K157001)
piRNA libraries: Imunoprecipitated RNA was ligated to 3` adapter containing unique indexes and two random bases (2N), and ligation products in 48 to 58-nt size range (equivalent to 19 - 29-nt long initial fragments) were recovered from Urea PAGE gel. Purified fragments were then ligated to 5` adapter containing eight random bases (8N) and purified on Urea PAGE gel again by selecting fragments in 82 - 92-nt size range. Final fragments were PCR amplified to add sequencing adapters. Total small RNAs libraries: A pellet of one million cells was used to purifiy small RNAs using PureLink miRNA Isolation kit (Thermo Fisher Scientific, K157001) and were spiked with a mix of 26 nt-long synthetic RNA callibrator sequences. RNA was ligated to 3` adapter containing unique indexes and two random bases (2N), and ligation products in 48 to 58-nt size range (equivalent to 19 - 29-nt long initial fragments) were recovered from Urea PAGE gel. Purified fragments were then ligated to 5` adapter containing eight random bases (8N) and PCR ampliffied to add sequencing adapters.

value: ovarian somatic sheet cells (OSC)

value: WT

value: GTTCAGAGTTCTACAGTCCGACGATCNNNNNNNN

value: NNTGACTGTGGAATTCTCGGGTGCCAAGG


title: m2_div6_ATACseq

tag: Sex


title: m2_div5_ATACseq

tag: sorting markers


title: m2_div4_ATACseq


title: m2_div1_ATACseq


title: BI102 Dye Swap

source_name: Ref

tag: tissue

value: Universal Reference

value: deceased

value: Oropharynx squamous cell carcinoma

value: Negative


title: tsRNA_1M_S6_RPI37


title: tsRNA_1M_S1_RPI12


title: 34192: keratinocytes ko-2


title: tsRNA_1M_S2_RPI13


title: tsRNA_1M_S5_RPI36


title: 34190: keratinocytes ko-1


title: tsRNA_1M_S4_RPI16


title: tsRNA_1M_S3_RPI14


title: 34191: keratinocytes wt-2

value: control


title: F7-4-8 [Bisulfite-Seq]

city: Copenhagen

first: Ali

last: Altintas

address: Blegdamsvej 3B

department: NNF Center for Basic Metabolic Research

country: Denmark

institute: Copenhagen University

data_processing:
1) Reads were trimmed using Trim Galore!  v0.4.3 with the --rrbs flag.
2) Trimmed reads were mapped to mm10 (GENCODE vM13) and methylation levels estimated using Bismark v0.18.1 and Bowtie2 v2.2.5 on default settings.
3) The SNPs (dbSNP build 142) with major allele frequency 0.95 at most in autosomal chromosomes were filtered out.
4) CpGs with coverage larger than 10 times the 95-percentile of coverage in each sample are filtered out.
5) Differential methylation analysis was performed by edgeR v3.24.0 using "(sequencing) pool" as a covariate for maternal and paternal lines separately. CpGs with minimum 8 counts were considered for differential methylation analysis.
Genome_build: mm10
Supplementary_files_format_and_content: Coverage (cov) files contain the coverage per sample (data processing step 2)

source_name: Quadricep

extract_protocol:
Allprep® DNA/RNA/miRNA Universal Kit (Qiagen) according to the manufacturer’s instructions
DNA samples were incubated with MpsI enzyme (NEB #R0106L). Bisulfite conversion was performed using the EZ DNA methylation lightening Kit (Zymo Research). The DNA was then PCR amplified and ligated to TruSeq (Illumina) sequencing adapters. Libraries were subjected to 75-bp single-end sequencing on NextSeq 2500 (Illumina ).

treatment_protocol:
Study 1: Female mice (C57BL/6, 8 weeks old) were subjected to either normal chow (NC) diet (n = 4) or 60% high-fat (HF) diet (Research Diets, NJ; n = 8) for 6 weeks. For mating, a lean male mouse (C57BL/6, 14 weeks old) was placed in the cage with 2 female mice. Once confirmed pregnant (by vaginal plug), the male mouse was removed and the female mice were housed individually in cages equipped with running wheels, which were locked for the sedentary groups. The groups were defined as normal chow, sedentary (NC-Sed), maternal high-fat diet, sedentary (M-HF-Sed) and maternal high-fat diet, exercise (M-HF-Ex).
Study 2: Male mice (C57BL/6, 8 weeks old) were subjected to either normal chow (NC) diet (n = 4) or 60% high-fat (HF) diet (Research Diets, NJ; n = 4) for 6 weeks. Male mice were then mated with lean female mice (C57BL/6, 14 weeks old) and once confirmed pregnant (by vaginal plug), the male mouse was removed, and the female mice were house individually with running wheels as described above. The groups were defined as normal chow, sedentary (NC-Sed), paternal high-fat diet, sedentary (P-HF-Sed) and paternal high-fat diet, exercise (P-HF-Ex).
In both studies, all groups were provided normal chow with sedentary activity from the day after birth and thereafter.  Adult offspring were humanely euthanized at 10 months of age and quadricep muscle collected. Study 1 and 2 were conducted in parallel allowing the NC-Sed group to serve as the control group for both studies.

tag: diet

value: high fat

tag: treatment

value: Ex

tag: parent

value: M

tag: group

value: MHE

tag: pool

value: A


title: 34189: keratinocytes wt-1

value: keratinocytes


title: 34193: keratinocytes wt-3


title: tsRNA_1M_S7_RPI38


title: PA conventionalized (wildtype) hindgut 1 [WT_H1]

city: Columbus

first: Aturo

last: Vera Ponce de Leon

state: Ohio

department: EEOB

institute: The Ohio State University

data_processing:
Quality check of all raw reads was completed by FastQC 0.11.7. Reads within a ‘phred’ quality score below 30 and Illumina adaptors were removed from sequences by TrimGalore 0.4.5  (--illumina -q 30 --retain_unpaired --paired parameters). To discard possible bacterial contamination in samples, only reads that mapped to the publicly-available P. americana genome. For this, high quality reads from each treatment were mapped against the P. americana genome ((19) PGRX00000000.1) using Hisat2 2.1.0 (60) with the following parameters -p 8 -x P.americana.genome.index -1 <Tissue>.<replicate>.1.fq -2 <Tissue>.<replicate>.2.fq -S <file>.sam. All ‘sam’ files were converted to ‘bam’ files with SAMtools 1.6
For this, high quality reads from each treatment were mapped against the P. americana genome (PGRX00000000.1) using Hisat2 2.1.0 with the following parameters -p 8 -x P.americana.genome.index -1 <Tissue>.<replicate>.1.fq -2 <Tissue>.<replicate>.2.fq -S <file>.sam. All ‘sam’ files were converted to ‘bam’ files with SAMtools 1.6
Properly paired mapped reads were retrieved by fastq tool from SAMtools with the following parameters SAMtools fastq -@ 8 -f 2 -1 <Tissue>.<replicate>.1.mapped.fq -2 <Tissue>.<replicate>.2.mapped.fq <file>.bam. Finally, these mapped paired reads were used for de-novo transcriptomic assembly of the P. Americana gut using Trinty 2.5.0 with the following parameters: Trinity --SS_lib_type RF --seqType fq --max_memory 900G--SS_lib_type RF --seqType fq --max_memory 900G--SS_lib_type RF --seqType fq --max_memory 900G –CPU 48 –full_cleanup. The completeness of the P. americana gut transcriptome assembly was estimated by the BUSCO v 3.2.0 (63) pipeline (-m trans -l insecta_odb9 -c 16  parameters)using the insecta_odb9 orthologue database.
For transcriptome annotation, similarities to known proteins were retrieved by BLASTX (90) (Trintiy transcripts) and BLASTP (Transdecoder protein coding translated sequences) searches to UniRef90 database and for conserved domains (pfam) by HMMER. All BLAST and HMMER tables were merged and sorted using the Trinotate pipeline. All transcripts with at least one annotation by Trinotate were manually retrieved and used to create a P. americana gut master annotation table. As a last quality filter, all transcripts with at least one bacterial hits annotation were manually removed from the assembled transcripts. These no-bacterial quality filter transcript assemblies were considered for further analysis.  Additionally, all transcripts were annotaded using blastx searches to all protein coding genes of pathways (20,AMPK,Chitin,DPP,GRH,Growthfactor,Hedgehog,Hippo,IMD,Insulin,JACKSTAT,JH,JNK,Notch,Pathway,PPO,Toll,TOR and Wingless) described in "The genomic and functional landscapes of developmental plasticity in the American cockroach paper" PMID: 29559629.
Differential expression analysis was performed with DESeq2 pipeline, briefly, treatments were classified depending tissue (midgut M or hindgut H) and germ-free, gnotobiotic or conventionalized conditions. Counts of RNA-seq reads from each treatment mapping to Trinity assembled genes and isoforms were quantified by Salmon v 0.9. Total “raw” transcript counts matrix was generated by tximport Bioconductor library in R. The software DESeq2 was used to detect differentially expressed isoforms and genes using as a contrast each bacterial treatment (germ-free, gnotobiotic and conventionalized) in the midgut and the hindgut. Isoforms/genes were considered differentially expressed if the adjusted p-value (Benjamini-Hochber [BH] multiple test correction) was less than or equal to 0.05 and an absolute fold-change above 1.5.
Genome_build: De-novo transcriptome assembly (trinity.fasta file)
Supplementary_files_format_and_content: transcriptome assembly in fasta format
Supplementary_files_format_and_content: Transcritpome annotation tab separate format
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
Supplementary_files_format_and_content: Matrix table with the FPKM normalized values by DEseq2 for every gene and every sample
Supplementary_files_format_and_content: Matrix table with DESeq2 values (log2FoldChange, p-value and p-adjust value) for every gene and every contrast
Supplementary_files_format_and_content: Annotations of P. americana transcripts involved in metabolic pathways described in PMID: 29559629

organism: Periplaneta americana

extract_protocol:
Total RNA from two different regions (midgut and hindgut) of P. americana guts (pools of 10 gut tissues) were obtained by the RiboZol™ RNA Extraction Reagent (VWR) and cleaned with the PureLink RNA Mini Kit (ThermoScientific) following the on-column DNase digestion kit.
Poly(A) mRNA were filtered using the NEBNext Poly(A) mRNA Magnetic Isolation Module according to the manufacture’s instruction (NEB).
cDNA libraries were generated with the Ultra II directional RNA library kit (NEB). All samples were sequenced using an Illumina HiSeq 4000 sequencer (Illumina) with an average of 20 million pair-end reads (2x150 bp) at The James Cancer Center sequencing facilities (The Ohio State University, Columbus OH USA).

value: bacterial conventionalized hindgut


title: PA gnotobiotic hindgut 3 [N_H3]

value: gnotobiotic hindgut


title: BI100 Dye Swap

tag: hpv status

tag: survival


title: m1_div6_ATACseq

value: Female

tag: genotype/variation

value: Cd19+/+ Irf4fl/fl

tag: tissues for b cell isolation and recovered from hosts

value: Spleen

tag: host

value: uMT

tag: stimulation

value: LPS

tag: days post-lps inoculation of host


title: F7-4-5 [Bisulfite-Seq]

value: B


title: PA gnotobiotic hindgut 1 [N_H1]


title: PA germ-free hindgut 3 [F_H3]

value: germ-free hindgut


title: BI102

value: Oropharynx squamous cell carcinoma

value: Negative

value: deceased

value: Universal Reference


title: m2_div3_ATACseq


title: BI100


title: PA gnotobiotic hindgut 2 [N_H2]


title: PA conventionalized (wildtype) hindgut 2 [WT_H2]


title: PA conventionalized (wildtype) hindgut 3 [WT_H3]


title: F7-4-4 [Bisulfite-Seq]

value: high fat

value: Ex

value: M

value: C


title: m2_div0_ATACseq


title: PA germ-free hindgut 2 [F_H2]


title: PA germ-free hindgut 1 [F_H1]


title: F7-4-1 [Bisulfite-Seq]

value: MHE


title: PA conventionalized (wildtype) midgut 2 [WT_M2]

value: bacterial conventionalized midgut


title: m1_div4_ATACseq

value: Female


title: PA conventionalized (wildtype) midgut 3 [WT_M3]


title: F7-2-4 [Bisulfite-Seq]

value: Sed

value: MHS

value: A


title: PA conventionalized (wildtype) midgut 1 [WT_M1]


title: PA gnotobiotic midgut 2 [N_M2]

value: gnotobiotic midgut


title: m1_div1_ATACseq

value: Cd19+/+ Irf4fl/fl

value: Spleen

value: uMT

value: LPS


title: PA gnotobiotic midgut 3 [N_M3]


title: m1_div3_ATACseq


title: PA gnotobiotic midgut 1 [N_M1]


title: PA germ-free midgut 3 [F_M3]

value: germ-free midgut


title: F7-1-4 [Bisulfite-Seq]


title: PA germ-free midgut 1 [F_M1]


title: PA germ-free midgut 2 [F_M2]


title: F7-1-3 [Bisulfite-Seq]


title: F7-2-1 [Bisulfite-Seq]

value: D


title: m1_div0_ATACseq


title: 3C-seq of cells with Smc-CC425 and parS334 (at -304kb) inactivated in exponential phase in SMG at 37°C [BSG3798_3C]

city: Lausanne

first: Anna

last: Anchimiuk

state: Vaud

address: UNIL-Sorge District

department: Department of Fundamental Microbiology

country: Switzerland

institute: University of Lausanne

data_processing:
ChIP-seq data was trimmed using Trimmomatic-0.39 (SE -phred33, ILLUMINACLIP:TruSeq3-SE:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36), mapped with bowtie2 using --very-sensitive-local setting, sorted with samtools and obtained .bam files were further imported to SeqMonk for Read Count Quantitation.
Sequencing data from 3C-seq experiments was demultiplexed, adapters trimmed, and PCR duplicates removed using custom scripts. If additional sequencing was performed, it is indicated in the sample name as '_reseq' and the data was pooled prior to mapping. Next, data was processed as described at https://github.com/axelcournac/3C_tutorial.
Removal of PCR duplicates based on the 20 first bp of the read containing a 6 nt random tag (home made script).
Iterative alignment, Min-leght=20, step=5, bowtie2 --very-sensitive-local
Filtering, Mapping quality=30, no ambiguous reads
Assignment to restriction fragment
Removal of un-informative events (uncuts, loops etc) as described in Cournac et al. BMC 2012.
3C-seq: binning at 10kb. ChIP-seq: binning at 1kb.
Genome_build: NC_000964 (1A700) or NC_022898 (PY79)
Supplementary_files_format_and_content: 422x422 (for 1A700 strains) and 404x404 (for PY79 strains) normalized contact matrix in txt format for the 3C-seq data. '_pooled' in processed data file name contains a matrix resulting from processing data from sample which was resequenced and both fastq files were combined. Read Count Quantitation and generation of Probe Report in .txt format with SeqMonk (used All Reads, Correct for total read count, Correcct for Milion Reads) for the ChIP-seq data.

organism: Bacillus subtilis

growth_protocol: strains were grown overnight in SMG, diluted to OD600 0.004 in fresh SMG and grown at 30 or 37°C until reached an OD600 between 0.022-0.026

extract_protocol:
3C-seq:
3C libraries were prepared as previously described (Marbouty et al., 2015). Minimal media (SMG) was used instead of LB. Briefly, cells were grown in 400 ml of SMG medium to exponential phase (OD = 0.022-0.030) and fixed with fresh formaldehyde (3% final concentration) for 30 minutes at RT, followed by 30 minutes at 4°C and quenched for 30 minutes with 0.25 M glycine at 4°C. Fixed cells were harvested by filtering, washed in fresh SMG, frozen in liquid nitrogen and stored at -80°C until further use.
For time-course experiments 2 l preculture was first grown until mid-exponential phase (OD = 0.022-0.030) and next, appropriate culture volumes were added to fresh pre-warmed SMG so that at given time points 2 x 200 ml of culture at mid-exponential could be collected (two technical replicates). The cultures were induced with 2 mM theophylline or 1 mM IPTG, depending on the promoter used, Ptheo or Pspank, respectively. Due to characteristics of the theophylline switch, the pre-culture as well as induction was performed at 30°C.
Frozen pellets were resuspended in 600 µl 1x TE and incubated at RT for 20 minutes with 4 µl of Ready-lyze lysozyme (35U/ul, Tebu Bio). Next, SDS was added to a final concentration of 0.5% and incubated at RT for 10 minutes.
50 µl of lysed cells were aliquoted to 8 tubes containing 450 µl of digestion mix (1x NEB 1 buffer, 1% triton X-100, and 100 U HpaII enzyme (NEB)) and incubated at 37°C for 3 hours with constant shaking. Digested DNA was collected by centrifugation, diluted into 4 tubes containing 8 ml of ligation mix (1x ligation buffer: 50 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT), 1 mM ATP, 0.1 mg/ml BSA, 125 U T4 DNA ligase 5 U/ml) and incubated at 16°C for 4 hours. Ligation reaction was followed by O/N decrosslinking at 65°C in the presence of 250 µg/ml proteinase K (Eurobio) and 5 mM EDTA.
DNA was precipitated with 1 volume of isopropanol and 0.1 Volume of 3 M sodium acetate (pH 5.2, Sigma) at -80°C for 1 hour. After centrifugation, the DNA pellet was resuspended in 1x TE at 30°C for 20 minutes. Next, DNA was extracted once with 400 µl phenol-chloroform-isoamyl alcohol solution and precipitated with 1.5 volume cold 100% ethanol in the presence of 0.1 volume 3 M sodium acetate at -80°C for 30 minutes. The pellet was collected and resuspended in 30 µl TE with RNaseA at 37°C for 30 minutes. All tubes were pooled and the resulting 3C library was quantified on gel using ImageJ.
ChIP-seq:
ChIP samples were prepared as described previously (Bürmann et al., 2017). Briefly, cells were cultured in 200 ml of minimal media (SMG) at 37°C until mid-exponential phase (OD=0.022-0.030) and fixed with buffer F (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.5 mM EGTA pH 8.0, 1 mM EDTA pH 8.0, 10% (w/v) formaldehyde) for 30 minutes at RT with occasional shaking. Cells were harvested by filtration and washed in PBS. Each sample was adjusted for 2 OD units (2 ml at OD600 = 1) and resuspended in TSEMS lysis buffer (50 mM Tris pH 7.4, 50 mM NaCl, 10 mM EDTA pH 8.0, 0.5 M sucrose and PIC (Sigma), 6 mg/ml lysozyme from chicken egg white (Sigma)). After 30 minutes of incubation at 37°C with vigorous shaking protoplasts were washed again in 2 ml TSEMS, resuspended in 1 ml TSEMS, split into 3 aliquots, pelleted and, after flash freezing, stored at -80°C until further use.
For time-course experiments 1 l preculture was first grown until mid-exponential phase (OD = 0.022-0.030) and next, appropriate culture volumes were added to fresh pre-warmed SMG so that at given time points 200 ml of culture at mid-exponential could be processed. The cultures were induced with 2 mM theophylline (Ptheo promoter). Due to characteristics of the theophylline switch, the pre-culture as well as induction was performed at 30°C.
For IP of samples for ChIP-seq, each pellet was resuspended in 1 ml of buffer L (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 1% (v/v) Triton X-100, 0.1% (w/v) Na-deoxycholate, 0.1 mg/ml RNaseA and PIC (Sigma)) and transferred to milliTUBE 1ml AFA Fiber and sonicated in a Covaris E220 water bath sonicator for 5 minutes at 4°C, 100 W, 200 cycles, 10% load and water level 5. Next, lysates were transferred into 2 ml tubes and centrifuged 10 minutes at 21000g at 4°C. 800 µl of supernatant was used for IP and 200 µl was kept as WCE (whole cell extract).
For IP, first, antibody serum was incubated with Protein G coupled Dynabeads (Invitrogen) in 1:1 ratio for 2.5 hours at 4°C with rotation. Next, beads were washed in buffer L and 50 µl were aliquoted to each sample tube. Samples were incubated for 2 hours at 4°C with rotation, followed by a series of washes with buffer L, buffer L5 (buffer L containing 500 mM NaCl), buffer W (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 1 mM EDTA pH 8.0) and buffer TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0). Finally, the beads were resuspended in 520 µl buffer TES (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1% (w/v) SDS). 300 µl of TES and 20 µl of 10% SDS were also added to WCE. Both tubes were incubated O/N at 65°C with vigorous shaking to reverse formaldehyde crosslinks.
Following rabbit polyclonal antibodies (manufactured by Eurogentec) were used:
For anti-scpB ChIP time-course experiment COD3 BsScpB-His6
For other anti-scpB ChIP experiments COD33 BsScpB-His6
For anti-parB ChIP experiments COD11 BsParB-His
Phenol-chloroform extraction was performed to purify the decrosslinked DNA. Samples were transferred to screw cap 1.5-ml tubes and first mixed vigorously with 500 µl of phenol equilibrated with buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). After centrifugation (10 minutes, RT, 13000 rpm), 450 µl of the aqueous phase was transferred to a new screw cap tube and mixed with equal volume of chloroform, followed by centrifugation. 400ul of aqueous phase was recovered for DNA precipitation with 2.5x volume of 100 % ethanol, 0.1x volume of 3M NaOAc and 1.2 µl of GlycoBlue and incubated for 20 minutes at -20°C. Next, samples were centrifuged for 10 minutes at 20000 g at RT and pellets obtained pellets were resuspended in 10 µl of EB (Qiagen) shaking at 55°C for 10 minutes and finally purified with a PCR purification kit, eluting in 50 uL EB.
3C-seq
1 ug of 3C library was suspended in water (final volume 130 µl) and sonicated using Covaris S220 (following manufacturers recommendations to obtain 500 bp target size). Next, DNA was purified with Qiagen PCR purification kit, eluted in 40 µl EB and quantified using NanoDrop. 1 ug of DNA was processed according to manufacturer instructions (Paired-End DNA sample Prep Kit – Illumina – PE-930-1001), except that DNA was ligated to custom-made adapters for 4 hours at RT, followed by inactivation step at 65°C for 20 minutes. DNA was purified with 0.75x AMPure beads and 3 µl were used for 50 µl PCR reaction (12 cycles). Amplified libraries were purified on Qiagen columns and pair end sequenced on an Illumina platform (HiSeq4000 or NextSeq).
ChIP-seq
For deep sequencing, the DNA libraries were prepared by Genomic Facility at CIG, UNIL, Lausanne. Briefly, the DNA was fragmented by sonication (Covaris S2) to fragment sizes ranging from 220–250 bp. DNA libraries were prepared using the Ovation Ultralow Library Systems V2 Kit (NuGEN) including 15 cycles of PCR amplification. Five to ten million single end sequence reads were obtained on a HiSeq2500 (Illumina) with 150-bp read length

tag: strain/genotype information


title: 3C-seq of cells with Smc-CC425 and parSopt at -304kb in exponential phase in SMG at 37°C [BSG3882_3C]


title: F6-3-4 [Bisulfite-Seq]

value: high fat

value: P

value: PHE