title: Sth1-AID + Rap1-AID degron induction RNA-Seq biological replicate 3

data_processing:
Adapter trimming was performed with cutadapt (version 1.9.1) (Martin M, 2011) with parameters "--minimum-length=25 --quality-cutoff=20 -a AGATCGGAAGAGC -A AGATCGGAAGAGC".
BWA (version 0.5.9-r16) (Li & Durbin, 2009) using default parameters was used to perform the read mapping independently to both the S. cerevisiae (assembly R64-1-1, release 90) and S. pombe (assembly ASM294v2, release 44) genomes.
Genomic alignments were filtered to only include those that were primary, properly paired, uniquely mapped, not soft-clipped, maximum insert size of 2kb and fewer than 3 mismatches using BamTools (version 2.4.0; (Barnett et al, 2011)).
Read counts relative to protein coding genes were obtained using the featureCounts tool from the Subread package (version 1.5.1) (Liao et al, 2014). The parameters used were “-O --minOverlap 1 --nonSplitOnly --primary -s 2 -p -B -P -d 0 -D 1000 -C --donotsort”.
Genome_build: Ensembl R64-1-1 release 90
Supplementary_files_format_and_content: Tab-delimited text file containing raw counts generated by featureCounts where gene names are rows and columns represent all samples generated in the study.

extract_protocol:
Cell pellets were washed once with sterile water and snap-frozen in liquid nitrogen. Together with fixed OD units of S. pombe cells as spike in, total RNA was extracted using the hot acid phenol protocol (acid phenol:chloroform:isoamyl alcohol 125:24:1) and TE-SDS buffer, then precipitated in ethanol with 0.3 M sodium acetate before re-suspension in DEPC-treated sterile water.
Total RNA from yeast was incubated with rDNase (Machery-Nagel rDNase Set) and column purified (Machery-Nagel NucleoSpin RNA) prior to sequencing library preparation. Total RNA was used to generate strand-specific libraries for polyadenylated RNA sequencing using the TruSeq Stranded mRNA Library Prep Kit (Illumina RS-122-2101) according to the manufacturer’s instructions. Library quality control was performed using the Agilent 2200 TapeStation with D1000 screentape (Agilent Technologies). Each library was sequenced on the HiSeq 2500 platform using V4 chemistry (Illumina) and typically generated ~55 million 101 bp strand-specific paired-end reads per sample.


title: D6_IMQ_Fos_CNR_Rep2

status: Public on Jul 27 2021

type: SRA

city: New York

first: Samantha

middle: B

last: Larsen

state: NY

address: 435 East 30th Street

country: USA

institute: NYU School of Medicine

description:
PCR amplified library from genomic DNA
D6_IMQ_Fos_CNR.peaks.bed (in CUTandRUN_bedfiles.tar.gz)

library_source: genomic

data_processing:
Library strategy: CUT&RUN-seq
Reads were aligned to reference genome (mm10) using Bowtie2 (version 2.2.9) and deduplicated with Picard tools (http://broadinstitute.github.io/picard, version 2.3.0). Reads were flittered to reads smaller or equal to 120bp using samtools (version 1.3.1). BAM files for each replicate were combined using samtools.  Bigwigs were generated using deeptools (version 3.1.2) with CPM (counts per million) normalization and presented by Integrative Genomics Viewer (IGV) software. Peaks were called using SEACR (Meers et al. 2019 ) using stringent setting and a numeric threshold of 0.01. Peaks were further filtered to have peaks scores greater than 0.5 for a set of high confident peaks per antibody and condition.
Genome_build: mm10

molecule: genomic DNA

organism: Mus musculus

source_name: skin

extract_protocol:
Cells were FACS sorted according to characteristics: FACS markers. 400,000 cells were resuspended in crosslinking buffer (10mM HEPES-NaOH pH 7.5, 100mM NaCl, 1mM EGTA, 1mM EDTA, 1% formaldehyde) and rotated at room temperature for 10 min. Crosslinked cells were quenched with glycine at a final concentration of 0.125M for 5 min at RT. Cells were washed with cold PBS and resuspended in NE1 buffer (20mM HEPES-KOH pH7.9, 10mM KCl, 1mM MgCl2, 1 mM DTT, 0.1% triton X-100 supplemented with Roche complete protease inhibitor EDTA-free) and rotated for 10 min at 4’C. Nuclei were washed twice with CUT&RUN wash buffer (20mM HEPES pH7.5, 150mM NaCl, 0.5% BSA, 0.5mM spermidine supplemented with protease inhibitor) and incubated with concanavalin-A (ConA) beads washed with CUT&RUN binding buffer (20mM HEPES-KOH pH 7.9, 10mM KCl, 1mM CaCl2, 1mM MnCl2) for 10 min at 4’C. ConA-bead-bound nuclei were incubated overnight at 4’C in CUT&RUN antibody buffer (CUT&RUN wash buffer supplemented with 0.1% triton X-100 and 2mM EDTA) and antibody. After antibody incubation, ConA-bead-bound nuclei were washed once with CUT&RUN triton wash buffer (CUT&RUN wash buffer supplemented with 0.1% triton X-100) then resuspended and incubated at 4’C for 1hr in CUT&RUN antibody buffer and 2.5uL pAG-MNase (EpiCypher). ConA-bound-nuclei were then washed twice with CUT&RUN triton wash buffer and resuspended in 100uL of lysis buffer (50mM Tris-HCl, 150mM NaCl, 1% triton X-100, 0.1% sodium deoxycholate, 5mM CaCl2) and incubated on ice for 30 min. The reaction was stopped by addition of 100ul 2x stop buffer (30mM EGTA) and incubated at 37’C for 10 min. After incubation, ConA-boun-nuclei were captured using a magnet and supernatant containing CUT&RUN DNA fragments was collected. Supernatant was incubated at 70’C for 2hrs with 2uL 10% SDS and 2.5uL 20mg/mL proteinase K. DNA was purified using PCI and overnight ethanol precipitation with glycogen at -20’C. DNA was resuspended in 15uL of buffer EB. CUT&RUN sequencing libraries were generated using NEBNext Ultra II DNA Library Prep Kit for Illumina and NEBNext Multiplex Oligos for Illumina (Index Primer Set 1 and 2). PCR-amplified libraries were purified using 1.2x ratio of AMPure XP beads and eluted in 15uL 0.1x TE buffer
NEBNext Ultra II DNA Library Prep Kit for Illumina

treatment_protocol: 2-3 postnatal day 60  murine backskin was treated with Imiquimod for 6 consecutive days

tag: Sex

value: female

tag: cell type

value: Epidermal Stem Cells

tag: strain

tag: genotype

value: wildtype

tag: facs markers

value: ITGA6+, ITGB1+, Sca1+, CD34-

tag: treatment

value: Imiquimod for 6 days


title: RamDA-seq iPS_C06

city: Tsukuba

first: Hiroaki

last: Tateno

state: Ibaraki

address: Tsukuba Central 2, 1-1-1 Umezono

country: Japan

institute: National Institute of Advanced Industrial Science and Technology (AIST)

library_source: transcriptomic

data_processing:
Library strategy: RamDA-seq
Quality check of reads in FASTQ files was performed using FastQC (version 0.11.5). The reads were trimmed by using fastq-mcf (version 1.0) with the parameters “-L 150 -l 50 -k 4.0 -q 30”. The trimmed reads were mapped to the human reference genome (GRCh38, primary assembly) using HISAT2 (version 2.1.0) with the parameters “--no-softclip”. The resulting SAM files were converted into BAM files using samtools (version 1.4). RSeQC (version 1.2) was used for quality check of read mapping. The featureCounts command in Subread (version 1.6.4) was used to quantify gene-level expression with the parameters “-M -O --fraction -p” and the reference gene model (GENCODE v32, primary assembly).
Genome_build: hg38
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

molecule: total RNA

organism: Homo sapiens

source_name: iPS cell

growth_protocol: 201B7 hiPSCs were cultured in 2 mL of mTeSR Plus (VERITAS ) on 6 well plate coated with Matrigel (Corning international ).

extract_protocol:
Single-cell cDNA library was prepared using GenNextⓇRamDA-seqTM Single Cell Kit (Toyobo Co., LTD. Tokyo, Japan) followed by manufacturer’s protocol.
cDNA library from each single cell was treated with NovaSeq 6000 S4 reagent Kit followed by manufacturer’s protocol.

treatment_protocol: 201B7 hiPSCs were cultured for 11 days using STEMdiff SMADi Neural Induction Kit (VERITAS) to differentiate into neural progenitor cells.

tag: cell line

value: iPS


title: Ctrl_1

city: 南京

first: Dandan

last: Zheng

state: 江苏

address: 南京医科大学江宁校区

country: China

institute: 南京医科大学

data_processing:
Using the Illumina MicroRNA sequencing
Raw reads were processed with ACGT101-miR (LC Sciences, Houston, Texas, USA) to remove adapter dimers, junk, low complexity reads, common RNA families (rRNA, tRNA, snRNA, and snoRNA) and repeats. Subsequently, unique sequences containing 18–26 bases were mapped to specific species precursors in miRBase 22.0 using a BLAST search to identify known miRNAs and novel miRNA-3p- and miRNA-5p-derived miRNAs. The mapped sequences were identified as known miRNAs. The remaining sequences were mapped to other selected species precursors in miRBase 22.0 by BLAST search. Differential expression of miRNAs as a function of normalized deep sequence counting was analyzed through the selective use of Student’s t-test. The significance threshold was set at <0.05.
The differentially expressed microRNAs were selected with log2 (fold change) >1 or log2 (fold change) <-1 and with statistical significance (p value < 0.05) by R package – Ballgown.
Genome_build: NOT PROVIDED
Supplementary_files_format_and_content: abundance measurements

growth_protocol: The human CRC cell lines HCT116 was purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. HCT116 cells were cultured in RPMI 1640 medium (HyClone, South Logan, UT, USA), supplemented with 10% FBS (Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Gibco). HCT116 cells were cultured at 37°C in a humidified incubator of 5% CO2.

extract_protocol:
Total RNA, including small RNAs from two groups of control and RANBP1-knockdown HCT116 cells, were extracted using the Total RNA Purification Kit from Norgen Biotek Corp (Thorold, Canada). Approximately 1 μg total RNA was used to prepare a small RNA library according to the protocol of the TruSeq Small RNA Sample Prep Kit (Illumina, San Diego, CA). Then, single-end sequencing (36 bp or 50 bp) was performed on the Illumina HiSeq 2500 System (LC Sciences, Houston, TX) according to the manufacturer’s recommended protocol.
MicroRNA sequencing Total RNA, including small RNAs, was extracted using a commercial extraction kit from Total RNA Purification Kit from Norgen Biotek Corp (Thorold, ON, Canada) in cultured mCECs in the SCIP assay. RNA quality and quantity were analyzed using a Bioanalyzer 2100 (Agilent, CA, USA) to ensure a RIN number > 7.0.

treatment_protocol: Human colon cancer cell line HCT116 was transfected with a control non-targeting shRNA to cells or transfected with shRNA targeting RANBP1 for 48 hours in RPMI medium.

tag: cell ine

tag: genotype/variation

value: control


title: starved HUVEC_exp 1

city: Amsterdam

first: Aldo

last: Jongejan

address: Meibergdreef 9

department: Epidemiology & Data Science

country: Netherlands

institute: Amsterdam University Medical Centers

description: rawCounts.txt

data_processing:
Alignment of sequence reads was performed for the individual samples against the GRCh38/hg38 reference genome using the HISAT2 aligner (v2.1.0) applying the default parameters and --dta-cufflinks --rna-strandness R
Counts were obtained using the corresponding genome annotation (v94) using HTSEq (v0.11) using the following parameters: -m union -f bam -r name -s reverse -a 10 -t exon -i gene_id
The obtained counts were used as input for the consecutive analysis using the statistical computing environment R (version 3.5.2, Bioconductor v3.8). Only features with CPM (counts per million mapped reads) of more than 2 in 3 or more samples were retained in the analysis (21502 features).
Counts were normalized by applying the trimmed mean of M-values method.
The count data was transformed to log2-counts per million (logCPM) using voom, estimating the mean-variance relationship.  Differential expression was assessed using a moderated t-test using the linear model framework from the limma package (v3.38.3).
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited file contains raw count data as obtained using HTSeq; rownames contain featurenames (ENSEMBL id); one column per sample.

source_name: HUVEC

growth_protocol: HUVECs were grown in endothelial growth medium (Promocell)

extract_protocol:
Cells were lysed in Trizol and RNA was extracted with RLT buffer
RNA libraries were prepared for sequencing using standard Illumina protocols. Kapa total RNA HyperPrep kit was used.

treatment_protocol: HUVECs were lentivirally transduced with scrambled sequences or pools of short hairpins targeting Rab5A or Rab5C, cloned in PLKO.1 (MISSION library) and next selected with puromycin for three days. Then they were replated, deprived of growth factors overniht and then stimulated or not with VEGF for the indicated time-points

value: primary endothelial cells from umbilical vein

tag: knockdown

tag: timepoint

value: 0 hr

tag: stimulation


title: RamDA-seq iPS_C07


title: Product, biological replicate 1, technical replicate 2

city: Gwanak

last: Baek

state: Seoul

address: Gwank 1

country: South Korea

institute: Seoul National University

description:
in vitro transcribed RNA
In vitro processing with the purified Microprocessor complex
Products_MP_only.csv
Products_set1_2

data_processing:
We utilized FASTX Toolkit (version 0.0.13.2; http://hannonlab.cshl.edu/fastx_toolkit/), cutadapt (version 2.3; Martin, 2011), and seqtk (version 1.2; https://github.com/lh3/seqtk) for sequence processing.
We trimmed bases after the first 70 bases using fastx_trimmer (-l 70), then applied quality filtering with fastq_quality_filter (-q 25 -p 80).
For input libraries, only intact reads were collected by checking 5′ and 3′ common sequences from forward and reverse reads, respectively (cutadapt -g GCCTATTCAGTTACAGCG/ GTTGCTAGCTTCAGTACG --trimmed-only --no-trim). We then trimmed the first 3 bases that were added to desynchronize sequencing cycles (cutadapt -u 3).
For cleavage product libraries, we trimmed Illumina 3′ or 5′ adapter sequence from the 3′ end of the forward or reverse reads, respectively, and filtered out the trimmed reads shorter than 40 nt (cutadapt -a TGGAATTCTCGGGTGCCAAGG/ GATCGTCGGACTGTAGAACTCTGAAC -m 40). Moreover, PCR duplicates were disposed using fastx_collapser.
We aligned the assembled reads to the pri-miRNA construct backbones using Bowtie2 (version 2.2.4; Langmead and Salzberg, 2012), allowing up to 2 mismatches (--local --score-min L,-16,2 --no-mixed --no-discordant --norc).
Genome_build: Pri-miRNA construct sequences
Supplementary_files_format_and_content: csv files were built by counting read counts for each fragment with the unique start and end position for each pri-miRNA.

source_name: In vitro processing

growth_protocol: DNA templates harboring 125 pri-miRNA sequences were commercially synthesized on a massive parallel synthesis platform (Celemics). Common sequences were added at the ends for PCR amplification. T7 promoter was also added at the 5′ end during the PCR amplification step. Using these templates, the minimal pri-miRNAs were in vitro transcribed by using the MEGAscript T7 Transcription Kit (Thermo Fisher Scientific).

extract_protocol:
RNAs were obtained by phenol extraction and ethanol precipitation.
The eluate was incubated with 0.5 μl of 10 μM 3′ adapter (Illumina, TruSeq Small RNA Library Prep Kit), which is modified with additional six degenerated bases at the 5′ end, at 70°C for 2 min. The 3′ adapter-ligated fragments were dissolved in 3 μl of TDW and incubated with 0.5 μl of 10 μM 5′ adapter (Illumina, TruSeq Small RNA Library Prep Kit), which is modified with additional six degenerated bases at the 3′ end, at 70°C for 2 min. The adapter-ligated fragments were reverse-transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) and RT primer (Illumina, TruSeq Small RNA Library Prep Kit). For Illumina sequencing, cDNA was amplified by PCR that utilized Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific) and PCR primers from TruSeq Small RNA Library Prep Kit (Illumina). Meanwhile, input substrates were also reverse-transcribed using custom RT primers and PCR-amplified using PCR primers from TruSeq Small RNA Library Prep Kit (Illumina).

treatment_protocol: 40 fmol of pri-miRNA substrate was incubated with 200 fmol of recombinant Microprocessor at 37°C for 1 h in the 10 μl buffer containing 500 mM NaCl, 50 mM Tris-HCl pH 7.5, 2 mM MgCl2, 1 mM DTT, 1 U/μl SUPERase·In RNase inhibitor (Thermo Fisher Scientific) and 200 ng/μl UltraPure BSA (Thermo Fisher Scientific).

tag: protocol

value: In vitro processing


title: Product, biological replicate 1, technical replicate 1

description:
in vitro transcribed RNA
In vitro processing with the purified Microprocessor complex
Products_MP_only.csv
Products_set1_1


title: Sth1-AID + Rap1-AID degron induction RNA-Seq biological replicate 2

city: London

first: Folkert

last: van Werven

address: 1 Midland Road

country: United Kingdom

institute: Francis Crick Institute

organism: Saccharomyces cerevisiae

source_name: yeast strains in small batch culture

growth_protocol: Cells were grown in yeast extract-peptone-dextrose (YPD) medium in conical flasks at 30 °C, shaking at 300 RPM in incubators.

treatment_protocol: Cells were either left untreated (wild-type samples), or treated during exponential growth (OD600 ~0.8) by adding 3-indole-acetic acid (3-IAA, Sigma-Aldrich I3750) to a final concentration of 500 μM from a 1 M stock dissolved in DMSO, or an equivalent volume of DMSO (vehicle).

tag: strain/background

value: FW7232 (MATa, his3D1, leu2D0, met15D0, ura3D0 LEU2::pGPD1-osTIR::LEU sth1::STH1-3V5-IAA7::KanMX6 rap1::RAP1-3V5-IAA7::KANMX6 rpb3::RPB3-3xFLAG::NATMX6) BY4741 derivative


title: D30_IMQ_Fos_CNR_Rep2

description:
PCR amplified library from genomic DNA
D30_IMQ_Fos_CNR.peaks.bed (in CUTandRUN_bedfiles.tar.gz)


title: D6_IMQ_CrePos_TamPos_YFPneg_Fos_CNR_Rep1

description:
PCR amplified library from genomic DNA
D6_IMQ_CrePos_TamPos_YFPneg_Fos_CNR.peaks.bed (in CUTandRUN_bedfiles.tar.gz)


title: D6_IMQ_Fos_CNR_Rep1


title: RamDA-seq iPS_C05


title: D6_Ctrl_Fos_CNR_Rep2

description:
PCR amplified library from genomic DNA
D6_Ctrl_Fos_CNR.peaks.bed (in CUTandRUN_bedfiles.tar.gz)

treatment_protocol: 2-3 postnatal day 60  murine backskin was treated with vanicream for 6 consecutive days

value: control vanicream for 6 days


title: Ctrl_2


title: Products, biological replicate 2

description:
In vitro processing with the purified Microprocessor and SRSF3
Products_MP_SRSF3.csv

treatment_protocol: 40 fmol of pri-miRNA substrate was incubated with 200 fmol of recombinant Microprocessor and 20 pmol of SRSF3 at 37°C for 1 h in the 10 μl buffer containing 500 mM NaCl, 50 mM Tris-HCl pH 7.5, 2 mM MgCl2, 1 mM DTT, 1 U/μl SUPERase·In RNase inhibitor (Thermo Fisher Scientific) and 200 ng/μl UltraPure BSA (Thermo Fisher Scientific).


title: D6_Ctrl_Fos_CNR_Rep1

treatment_protocol: 2-3 postnatal day 60 mice backskin were treated with vanicream for 6 consecutive days


title: RamDA-seq iPS_C04


title: Sth1-AID + Rap1-AID degron no induction RNA-Seq biological replicate 3

value: DMSO


title: RamDA-seq iPS_C03


title: Products, biological replicate 1


title: D30_IMQ_Stat3KO_ATACseq_Rep2

description: PCR amplified library from genomic DNA

data_processing:
Reads were aligned to the reference genome (mm10) with Rsubread's align method in paired-end mode as descrived in doi::10.1093/nar/gkz114. Peaks were called with MACS2 software in BAMPE mode.
Genome_build: mm10

extract_protocol:
Cells were FACS sorted according to characteristics: FACS markers; Nuclei were extracted and genomic DNA was transposed per protocol outlines in doi::10.1038/nmeth.2688
Nextera DNA Library Preparation Kit

treatment_protocol: Topically treated with 200 ul 1 mg/mL 4-hydoxytamoxifen for 3 consecutive days beginning at P46. Imiquimod treatment for 6 days beginning at P60. 2-3 mice pooled.

value: YFP+, "ITGA6+, ITGB1+, Sca1+, CD34-

value: 4-hydroxytamoxifen treated, Imiquimod for 6 days


title: Sth1-AID + Rap1-AID degron no induction RNA-Seq biological replicate 2


title: Input, biological replicate 2

source_name: In vitro transcription

value: In vitro transcription


title: Sth1-AID + Rap1-AID degron induction RNA-Seq biological replicate 1


title: D30_IMQ_Stat3KO_ATACseq_Rep1

description: PCR amplified library from genomic DNA


title: RamDA-seq iPS_B10


title: Sth1-AID + Rap1-AID degron no induction RNA-Seq biological replicate 1


title: D30_Ctrl_Stat3KO_ATACseq_Rep2

description: PCR amplified library from genomic DNA

treatment_protocol: Topically treated with 200 ul 1 mg/mL 4-hydoxytamoxifen for 3 consecutive days beginning at P46. Vanicream treatment for 6 days beginning at P60. 2-3 mice pooled.

value: 4-hydroxytamoxifen treated, control vanicream for 6 days


title: Rap1-AID degron induction   RNA-Seq biological replicate 2

value: FW7238 (MATa, his3D1, leu2D0, met15D0, ura3D0 LEU2::pGPD1-osTIR::LEU rap1::RAP1-3V5-IAA7::KANMX6 rpb3::RPB3-3xFLAG::NATMX6) BY4741 derivative


title: Rap1-AID degron induction  RNA-Seq biological replicate 3


title: PC3 proL1 2 C

city: Bronx

first: Kelvin

middle: Paul

last: Davies

state: New York

address: 1300 Morris Park Avenue, Forchheimer 742

department: Urology

institute: Albert Einstein College of Medicine

description:
Transformed to overexpress proL1 via lentiviral transduction
DESeq2_normalized_count_matrix.txt

data_processing:
Reads were aligned with STAR (v 2.4.0c)
Genes were annotated in Gencode v18
Reads were quantified with featureCounts (v 1.4.3-p1)
Normalization and differential experession was performed with Bioconductor package DESeq2
Genome_build: GRCh37.p12
Supplementary_files_format_and_content: Tab-delimited text files include count matrix for counts per gene, normalized for library size using DESeq2 (*DESeq2_count_matrix.txt) and count data transformed to the log2 scale in a way which minimizes differences between samples for rows with small counts, and normalizes with respect to library size (*DESeq2_regularized_log_transformed.txt)

source_name: Prostate cancer cells

growth_protocol: PC3 cells were routinely passaged every 7 days (typically 2-3 x 10^6 cells into a T75 flask) in RPMI 1640 supplemented with 10% FBS, 100 units/mL penicillin, and 0.1 mg/mL streptomycin

extract_protocol:
RNA was harvested using the Qiagen RNeasy Mini Kit and its quality was checked using an Agilent 2100 Bioanalyzer
Illumina TruSeq Stranded Total RNA Library Prep, with Ribosomal Depletion

value: proL1 overexpression


title: RamDA-seq iPS_B07


title: D6_IMQ_Stat3KO_ATACseq_Rep2

description: PCR amplified library from genomic DNA

value: female

value: Epidermal Stem Cells


title: D30_Ctrl_Stat3KO_ATACseq_Rep1

description: PCR amplified library from genomic DNA


title: PC3 proL1 3 C


title: PC3 proL1 1 C


title: RamDA-seq iPS_A10


title: Rap1-AID degron no induction RNA-Seq biological replicate 3


title: RamDA-seq iPS_A08


title: RamDA-seq iPS_B04


title: PC3 wt 3 C

value: parental cell line


title: RamDA-seq iPS_A07

value: iPS


title: Rap1-AID degron induction RNA-Seq biological replicate 1


title: D6_IMQ_Stat3KO_ATACseq_Rep1

description: PCR amplified library from genomic DNA


title: Rap1-AID degron no induction RNA-Seq biological replicate 2


title: Rap1-AID degron no induction RNA-Seq biological replicate 1


title: D6_Ctrl_Stat3KO_ATACseq_Rep2

description: PCR amplified library from genomic DNA


title: PC3 wt 2 C


title: RamDA-seq iPS_A06


title: D6_IMQ_CreNeg_TamPos_ATACseq_Rep1

description: PCR amplified library from genomic DNA

value: ITGA6+, ITGB1+, Sca1+, CD34-


title: PC3 wt 1 C


title: Input, biological replicate 1


title: D6_Ctrl_Stat3KO_ATACseq_Rep1

description: PCR amplified library from genomic DNA


title: RamDA-seq iPS_A05


title: RamDA-seq iPS_A04


title: Sth1-AID degron induction RNA-Seq biological replicate 3

value: FW7220 (MATa, his3D1, leu2D0, met15D0, ura3D0 LEU2::pGPD1-osTIR::LEU sth1::STH1-3V5-IAA7::KanMX6 rpb3::RPB3-3xFLAG::NATMX6) BY4741 derivative


title: 760-3_Downhill Run corrected-delEx44 DMD

city: Dallas

first: Jiwoong

last: Kim

state: Texas

address: 5323 Harry Hines Blvd.

institute: UT Southwestern Medical Center

data_processing:
Trim Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) was used for quality and adapter trimming. The mouse reference genome sequence and gene annotation data, mm10, were downloaded from Illumina iGenomes (https://support.illumina.com/sequencing/sequencing_software/igenome.html). The qualities of RNA-sequencing libraries were estimated by mapping the reads onto mouse transcript and ribosomal RNA sequences (Ensembl release 89) using Bowtie (v2.3.4.3) (Langmead and Salzberg, 2012). STAR (v2.7.2b) (Dobin et al., 2013) was employed to align the reads onto the mouse genome, SAMtools (v1.9) (Li et al., 2009) was employed to sort the alignments, and HTSeq Python package (Anders et al., 2015) was employed to count reads per gene. edgeR R Bioconductor package (Gentleman et al., 2004; Robinson et al., 2010; McCarthy et al., 2012) was used to normalize read counts and identify differentially expressed (DE) genes. KEGG (Kanehisa et al., 2017) pathway data was downloaded using KEGG API (https://www.kegg.jp/kegg/rest/keggapi.html) and gene ontology (GO) data was downloaded from NCBI FTP (ftp://ftp.ncbi.nlm.nih.gov/gene/DATA/gene2go.gz). The enrichment of DE genes to pathways and GOs were calculated by Fisher's exact test in R statistical package.
Genome_build: mm10
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

molecule: polyA RNA

source_name: Quadriceps Muscle

extract_protocol:
Quadriceps muscle was harvested and flash frozen in liquid nitrogen. RNA was extracted using miRNAeasy mini kit (Cat#217004).
Libraries were constructed using the Illumina TruSeq RNA Sample Prep Kit.

treatment_protocol: DelEx44 DMD mice injected intraperitoneally with 8 x 10^13 vg/kg each of scAAV9-sgRNA and ssAAV9-SpCas9 on day P4. At one month of age, tissue was either harvested or mice were run downhill. Mice were run at a 15 degree decline at a speed of 12 m/min every other day for 1 month.

tag: tissue

value: Quadriceps Muscle

tag: age

value: 8 Weeks

value: CRISPR-Cas9 treated delEx44


title: D6_Ctrl_CreNeg_TamPos_ATACseq_Rep2

description: PCR amplified library from genomic DNA


title: 760-1_Downhill Run corrected-delEx44 DMD


title: RamDA-seq iPS_A03


title: D6_Ctrl_CreNeg_TamPos_ATACseq_Rep1

description: PCR amplified library from genomic DNA


title: WT-2_Downhill Run WT

value: WT


title: Sth1-AID degron induction RNA-Seq biological replicate 1


title: D30_PI_AFOS_Dox_ATACseq_Rep2

description: PCR amplified library from genomic DNA

treatment_protocol: Imiquimod for 6 days at postnatal day P60 + Doxycline diet beginning at postnatal day P76, 2-3 mice pooeld

value: male

value: Pgk-H2B-RFP;TRE-AFOS K14rtTA

value: RFP+,ITGA6+, ITGB1+, Sca1+, CD34-

value: Imiquimod for 6 days at P60 + Doxycline diet beginning at P76


title: P2L_72h_24

city: Wuhan

first: Dong

last: Chen

state: Hubei

address: 388 GaoXin 2nd Road, East Lake Hi-Tech Development Zone

department: Center for Genome Analysis

institute: ABLife, Inc.

description: single embryo for total RNA-seq

data_processing:
bcl2fastq(v2.16.0.10) were used for base calling
Raw reads were first discarded if containing more than 2-N bases, then reads were processed by clipping adaptor, removing low quality bases, and discarding too short reads (less than 16nt). FASTX-Toolkit (Version 0.0.13) was used to get the clean reads.
After that, clean reads were aligned to the Honeybee genome (Amel 4.5) by tophat2 (Kim et al., 2013). Aligned reads with more than one genome location were discarded due to their ambiguous location.
Uniquely localized reads were used to calculate reads number and RPKM value (RPKM represents reads per kilobase and per million) for each gene according to reads and genes genome location. Other statistical results, such as gene coverage and depth, reads distribution around start codon and stop codon, were also obtained.
other analysis, such as gene expression differences ,were also performed.
Genome_build: Amel_4.5
Supplementary_files_format_and_content: expressed gene Reads & RPKM files, Microsoft excel format

organism: Apis mellifera

source_name: embryo

growth_protocol: The honeybee, Apis mellifera, colonies were maintained at the Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing, China, according to standard beekeeping technique. To control the egg laying, a mated queen was confined on an egg-free frame for two hours before placed back into the hive. Eggs were collected at 24 h (Day 1), 48 h (Day 2) and 72 h (Day 3) after laying, and 3 eggs were sampled for each day.

extract_protocol:
Worker and drone embryos were collected from the distinct type of frame laid by the same queen. We sampled two groups of worker and drone embryos laid by two different honeybee queens. Then a total of 36 embryos were collected for single-embryo RNA-seq.
High-quality, full-length cDNA is generated directly from 1–1,000 cells by The SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Cat. Nos. 634890). cDNA was amplified by LD PCR. RNA can be transcribed by T7 promoter from ds cDNA , RNA was treated with RQ1 DNase (promega) to remove DNA .The quantity of the purified DNA were determined by Qubit. For each sample, 200 ng RNA was used for RNA-seq library preparation. RNAs were iron fragmented at 95℃ followed by end repair and 5' adaptor ligation. Then reverse transcription was performed with RT primer harboring 3' adaptor sequence and randomized hexamer. The cDNAs were purified and amplified and PCR products corresponding to 200-500 bps were purified, quantified and stored at -80 ℃ until used for sequencing.

value: drone embryo

tag: developmental stage

value: embryonic development 72h

tag: batch

value: 2nd batch


title: WT-1_Downhill Run WT


title: Sth1-AID degron no induction RNA-Seq biological replicate 3

value: DMSO


title: D30_PI_AFOS_Dox_ATACseq_Rep1

description: PCR amplified library from genomic DNA

tag: Sex

value: female

tag: cell type

value: Epidermal Stem Cells


title: P2L_72h_23

description: single embryo for total RNA-seq


title: Cerebral Cortex_CVI_LCMV_Rep4

city: Bethesda

first: Kory

middle: R

last: Johnson

state: MD

address: 10/3B01, 9000 Rockville Pike

department: DIR IT & Bioinformatics

institute: NINDS/NIH

data_processing:
RTA 1.13.48 and CASAVA 1.8.2. software used for basecalling and deplexing.
Post deplexing, the paired-end sequence files (.fastq) per sample were quality inspected using the FastQC tool 0.11.8 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) then adaptor clipped (TruSeq3-PE-2.fa:2:30:10) and trimmed to remove 5' nucleotide bias (HEADCROP:11) and low quality calls (TRAILING:20 SLIDINGWINDOW:4:20 MINLEN:15) using the Trimmomatic tool 0.39 (http://www.usadellab.org/cms/?page=trimmomatic). Surviving intact pairs of reads per sample were then imported into the CLCbio Genomics Workbench 11 (https://www.qiagenbioinformatics.com/) and reference mapped by sample in stranded fashion against the current instance of the mouse genome (GRCm38) using the "RNA-Seq Analysis" tool supported therein under default parameters. Expression per known annotated gene (Mus_musculus.GRCm38.83.chr.gtf) in Transcripts Per Kilobase Million (TPM) units was then exported from the Workbench.
Genome_build: GRCm38
Supplementary_files_format_and_content: Tab-delimited text files containing enumerated expression per sample sequenced.

source_name: Cerebral cortex

extract_protocol:
At day 20 following injury, mice were perfused with normal saline, and a 3 x 3 x 3 mm cube of injured tissue from the Cerebral cortex was harvested and snap frozen using dry ice. The tissues were stored at -80oC until use. Total RNA was extracted with Qiagen kit following the manufacturer’s protocol, and RNA quantity and integrity was assessed by the Bioanalyzer (Agilent, Santa Clara, CA).
500 ng of total RNA was used in conjunction with the TruSeq® Stranded Total RNA Library Prep kit (Illumina, San Diego, CA). The libraries quality was checked by the Bioanalyzer and quantitated by the Qubit (ThermoFisher Scientific). Equimolar quantities from each sample library was pooled and run on a Highoutput Next-Seq550 kit.

treatment_protocol: B6 mice were divided into 3 groups, 4 mice per group: uninjured mice (Ctrl), naïve cerebrovascular injured mice (CVI), cerebrovascular injured mice infected with lymphocytic choriomeningitis virus (CVI LCMV).

value: Cerebral cortex

tag: sample group

tag: technical replicate

value: No

tag: biological replicate

value: Yes

tag: strain

tag: gender

value: Male

value: 13 Weeks


title: Sth1-AID degron no induction RNA-Seq biological replicate 2

value: FW7220 (MATa, his3D1, leu2D0, met15D0, ura3D0 LEU2::pGPD1-osTIR::LEU sth1::STH1-3V5-IAA7::KanMX6 rpb3::RPB3-3xFLAG::NATMX6) BY4741 derivative

tag: treatment


title: P2L_72h_17

description: single embryo for total RNA-seq


title: Sth1-AID degron induction RNA-Seq biological replicate 2


title: D30_Ctrl_AFOS_Dox_ATACseq_Rep2

description: PCR amplified library from genomic DNA

treatment_protocol: Vanicream for 6 days at postnatal day P60 + Doxycline diet beginning at postnatal day P76, 2-3 mice pooeld

tag: genotype

tag: facs markers

value: Vanicream for 6 days at P60 + Doxycline diet beginning at P76


title: Sth1-AID degron no induction RNA-Seq biological replicate 1


title: P2S_72h_22

description: single embryo for total RNA-seq

value: worker embryo


title: Cerebral Cortex_CVI_LCMV_Rep3


title: D30_Ctrl_AFOS_Dox_ATACseq_Rep1

description: PCR amplified library from genomic DNA

treatment_protocol: Vanicream for 6 days at postnatal day 60 + Doxycline diet beginning at postnatal day 76, 2-3 mice pooled

value: Pgk-H2B-RFP;TRE-AFOS K14rtTA

value: Vanicream for 6 days at P60 + Doxycline diet beginning at P76 +


title: Wild-type RNA-Seq biological replicate 3

value: FVW7228 (MATa, his3D1, leu2D0, met15D0, ura3D0 rpb3::RPB3-3xFLAG::NATMX) BY4741 derivative

value: no treatment


title: Cerebral Cortex_CVI_LCMV_Rep2


title: D6_IMQ_AFOS_Dox_ATACseq_Rep2

description: PCR amplified library from genomic DNA

treatment_protocol: doxycycline feed started on postnatal day 46, Imiquimod for 6 days starting on postnatal day 60, 2-3 mice

value: male

value: RFP+,ITGA6+, ITGB1+, Sca1+, CD34-

value: Doxycline diet beginning at P46 + Imiquimod for 6 days


title: P2S_72h_29

description: single embryo for total RNA-seq

value: embryonic development 72h

value: 2nd batch


title: P2S_72h_25

description: single embryo for total RNA-seq


title: Wild-type RNA-Seq biological replicate 2


title: Wild-type RNA-Seq biological replicate 1


title: D6_IMQ_AFOS_Dox_ATACseq_Rep1

description: PCR amplified library from genomic DNA

value: female

value: Epidermal Stem Cells


title: P1L_72h_11

description: single embryo for total RNA-seq

value: drone embryo

value: 1st batch


title: Cerebral Cortex_CVI_LCMV_Rep1

value: Cerebral cortex

value: No

value: Yes

value: Male

value: 13 Weeks


title: Cerebral Cortex_CVI_Rep3


title: P1L_72h_4

description: single embryo for total RNA-seq


title: D6_IMQ_AFOS_NoDox_ATACseq_Rep1

description: PCR amplified library from genomic DNA

treatment_protocol: Imiquimod for 6 days starting on postnatal day 60, 2-3 mice

value: Pgk-H2B-RFP;TRE-AFOS K14rtTA

value: Imiquimod for 6 days


title: 20_Resting_aDP_rep2

city: Los Angeles

first: Ping

last: Wu

state: CA

address: 2011 Zonal Ave HMR313A

department: Pathology

institute: University of Southern California

description: Apical dermal papilla from Resting phase rep2

data_processing:
RNA-seq analysis. The RNA-seq analysis pipeline was adapted according to the publication (Trapnell et al., 2012). Manipulation. Tools: fastq_groomer (Galaxy Version 1.1.1) and fastq_trimmer_by_quality (window size 3; min score >= 20). Mapping. Tool: TopHat2 (Galaxy Version 2.1.1). Quantification and differentially expressed gene analysis. Tools: Cufflinks package (Trapnell et al., 2010)–including Cufflinks (Galaxy Version 2.2.1.2), Cuffmerge (Galaxy Version 2.2.1.1), Cuffquant (Galaxy Version 2.2.1.1), and Cuffdiff (Galaxy Version 2.2.1.3).
Supplementary_files_format_and_content: TPM

organism: Gallus gallus

source_name: Adult contour feather (apical dermal papilla from Resting phase)

growth_protocol: 1 year old adult male white leghorn chicken

extract_protocol:
RNA extraction for RNA-seq: Total RNAs of indicated tissues were extracted followed by the standard Trizol extraction protocol (Invitrogen, Cat.15596026).
TruSeq RNA Prep Kit v2 (Illumina, RS-122-2001/-2002).

treatment_protocol: No treatment

tag: breed

value: white leghorn

tag: group


title: Cerebral Cortex_CVI_Rep2


title: P1L_72h_6

description: single embryo for total RNA-seq


title: 19_Resting_aDP_rep1

description: Apical dermal papilla from Resting phase rep1

value: male


title: Cerebral Cortex_CVI_Rep1


title: D6_IMQ_AFOS_NoDox_ATACseq_Rep2

description: PCR amplified library from genomic DNA

value: RFP+,ITGA6+, ITGB1+, Sca1+, CD34-


title: Cerebral Cortex_Ctrl_Rep4

description: uninjured

value: uninjured


title: 17_PP_NR_rep1

description: Pulp from Near Resting phase rep1

source_name: Adult contour feather (pulp from Near Resting phase)

value: Pulp from Near Resting phase


title: D6_Ctrl_AFOS_NoDox_ATACseq_Rep1

description: PCR amplified library from genomic DNA

treatment_protocol: control vanicream for 6 days starting on postnatal day 60, 2-3 mice

value: control vanicream for 6 days


title: D6_Ctrl_AFOS_Dox_ATACseq_Rep2

description: PCR amplified library from genomic DNA

treatment_protocol: doxycycline feed started on postnatal day 46, control vanicream for 6 days starting on postnatal day 60, 2-3 mice

value: Doxycline diet beginning at P46 + control vanicream for 6 days


title: Cerebral Cortex_CVI_Rep4


title: 15_PP_LG_rep1

description: Pulp from Late Growth phase rep1

source_name: Adult contour feather (pulp from Late Growth phase)

value: Pulp from Late Growth phase


title: P1S_72h_14

description: single embryo for total RNA-seq

value: worker embryo

value: embryonic development 72h


title: D6_Ctrl_AFOS_Dox_ATACseq_Rep1

description: PCR amplified library from genomic DNA

value: female

value: Epidermal Stem Cells


title: P2L_48h_26

description: single embryo for total RNA-seq

value: drone embryo

value: embryonic development 48h

value: 2nd batch


title: 18_PP_NR_rep2

description: Pulp from Near Resting phase rep2


title: P1S_72h_4

description: single embryo for total RNA-seq

value: 1st batch


title: 21_Resting_aDP_rep3

description: Apical dermal papilla from Resting phase rep3


title: D6_Ctrl_AFOS_NoDox_ATACseq_Rep2

description: PCR amplified library from genomic DNA

value: Pgk-H2B-RFP;TRE-AFOS K14rtTA


title: Cerebral Cortex_Ctrl_Rep3

description: uninjured

value: Cerebral cortex

value: No

value: Yes

value: Male

value: 13 Weeks


title: 16_PP_LG_rep2

description: Pulp from Late Growth phase rep2

value: white leghorn

value: male


title: P2L_48h_21

description: single embryo for total RNA-seq


title: Cerebral Cortex_Ctrl_Rep2

description: uninjured

value: uninjured


title: 11_ICRF_young

city: Vienna

first: Bart

middle: Johan H

last: Dequeker

address: Dr. Bohr-Gasse 3

country: Austria

institute: IMBA - Institute of Molecular Biotechology

data_processing:
Base calling was performed by VBCF NGS facility and NGS facility in the Department of Totipotency, MPIB
Mapping: iterative mapping (see Imakaev et al, 2012) using bwa
Read filtering was performed as in Flyamer et al., 2017:
Summary of filtering: If two reads map to the same strand, and each side of the read is within 500 bp of any side of the other read, we retain only one copy of the read. We separate the genome into 500 bp bins. If any two bins are interacting more than once, only one interaction is counted. Since each region is only present in the single cell up to 4 times (in oocytes), we eliminated all 500 bp bins that form more than 8 unique interactions with other bins.
Filtered reads were then binned at different resolutions (we provide 4 resolutions) using read start as a read position and stored in the Cool file format. No normalization was applied to the data.
Genome_build: mm9
Supplementary_files_format_and_content: Matrix of whole-genome locus interaction frequencies binned at 1kb, 10kb, 40kb, and 100kb resolutions stored in the Cool file format.

source_name: oocyte

extract_protocol:
snHi-C protocol according to Flyamer et al., 2017
The library preparation was performed following the standard DNA sample preparation protocol by the VBCF NGS unit or the NGS facility in the Department of Totipotency, MPIB

value: 2-2.5 month old (young)

value: GV oocyte - SN


title: Enhancer - barcode assignmennt CHEQ-seq SOX-MITF combinations

city: Leuven

first: Gert

last: Hulselmans

address: Herestraat 49 PO Box 602

department: Center for Brain and Disease Research

country: Belgium

institute: VIB

description:
processed data file:
CHEQ-seq_SOX-MITF_combinations_enh-BC_list.tsv.gz

data_processing:
Library strategy: CHEQ-seq
Enhancer - barcode assignment files are processed with a custom script. Enhancers and barcodes are separated with cutadapt. Enhancers are mapped to the human hg19 genome using minimap.
Raw cDNA and plasmid reads are first processed with a custom script to extract and count each barcode before associating them to the corresponding enhancer based on the enhancer - bc assignment list.
Enhancer activity from MPRA assay is estimated via a custom R script. Enhancers are first filtered based on the number of BCs identified in the sequencing reads. Thresholds of 5 (for the H3K37ac library and enhancer tiling library), 10 (for ATAC-seq library) or 20 (for the SOX-MITF combinations library) BCs per enhancer are selected based on the complexity of the library and the sequencing saturation of the enhancer - BC assignment samples. For the remaining enhancers, BC counts are aggregated per enhancer and then a count per million (CPM) normalisation is applied. Plasmid (input) and cDNA (output) samples are merged by keeping only enhancers remaining in both samples after filtering. Input normalisation is done by dividing CPM normalised cDNA counts by CPM normalised plasmid counts resulting in a FC value. For libraries with shuffled sequences, a basal expression normalisation is further applied by dividing the FC value of the enhancer by the median FC value of the shuffled sequences. For CHEQ-seq SOX10-KD library, enhancer activity from cDNA and plasmid barcode counts is performed with DEseq2.
Genome_build: hg19
Supplementary_files_format_and_content: Enhancer to barcode assignment lists are tables containing two columns: Enhancer name; barcode associated to that enhancer. Enhancer activity matrices are table containing: Enhancer name; Enhancer class; Enhancer activity Fold Change (one column per cell line). DEseq2 result table contains the following columns: Enhancer name; baseMean; log2FoldChange; lfcSE; stat; pvalue; padj; enhancer class.
CHEQ-seq_5p_H3K27ac_enh-BC_list.tsv: Enhancer to barcode assignment list
CHEQ-seq_Intron_H3K27ac_enh-BC_list.tsv: Enhancer to barcode assignment list
CHEQ-seq_ATAC_enh-BC_list.tsv.gz: Enhancer to barcode assignment list
CHEQ-seq_Enhancer-tiling-A_enh-BC_list.tsv.gz: Enhancer to barcode assignment list
CHEQ-seq_Enhancer-tiling-B_enh-BC_list.tsv.gz: Enhancer to barcode assignment list
CHEQ-seq_SOX10-KD_enh-BC_list.tsv: Enhancer to barcode assignment list
CHEQ-seq_SOX-MITF_combinations_enh-BC_list.tsv.gz: Enhancer to barcode assignment list
CHEQ-seq_5p_H3K27ac_processed.tsv: Enhancer activity matrix for CHEQ-seq 5' H3K27ac
CHEQ-seq_Intron_H3K27ac_processed.tsv: Enhancer activity matrix for CHEQ-seq Intron H3K27ac
CHEQ-seq_ATAC_processed.tsv: Enhancer activity matrix for CHEQ-seq ATAC
STARR-seq_ATAC_processed.tsv: Enhancer activity matrix for STARR-seq ATAC
CHEQ-seq_Enhancer_tiling_A_processed.tsv: Enhancer activity matrix for CHEQ-seq Enhancer tiling A
CHEQ-seq_Enhancer_tiling_B_processed.tsv: Enhancer activity matrix for CHEQ-seq Enhancer tiling B
CHEQ-seq_SOX10-KD_processed.tsv: DEseq2 result table for CHEQ-seq SOX10-KD
CHEQ-seq_SOX-MITF_combinations_processed.tsv: Enhancer activity matrix for CHEQ-seq SOX - MITF combinations

organism: synthetic construct

source_name: Plasmid

growth_protocol: Melanoma cell lines were cultured with Ham's F-10 nutrient mix + 10% FBS + 0.5% penicillin/streptomycin in an incubator at 37°C with 5% CO2.

extract_protocol:
Total RNA was extracted from the cell with the innuPREP RNA Mini Kit 2.0 (Analytik Jena) , followed by mRNA isolation using the Dynabeads mRNA purification kit (Ambion). cDNA synthesis was performed with the GoScript RT kit and oligo dT primer (Promega). DNA was extracted with a plasmid extraction protocol from Qiagen.
Plasmid libraries, extracted plasmids and cDNA were prepared for sequencing with two rounds of PCR. The first PCR targets the random barcode present within the transcript. The second PCR adds sequencing indexes to the PCR product.

tag: molecule subtype

value: Plasmid


title: P1S_72h_15

description: single embryo for total RNA-seq

tag: tissue

value: worker embryo

value: embryonic development 72h


title: P2L_48h_29

description: single embryo for total RNA-seq

value: drone embryo

value: embryonic development 48h


title: 10_ICRF_young

tag: age


title: 14_PP_EG_rep2

description: Pulp from Early Growth phase rep2

source_name: Adult contour feather (pulp from Early Growth phase)

value: Pulp from Early Growth phase


title: 13_PP_EG_rep1

description: Pulp from Early Growth phase rep1


title: 7_ICRF_young

tag: developmental stage


title: 11_cPP_rep2

description: Central pulp rep2

source_name: Adult contour feather (central pulp)

value: Central pulp


title: Enhancer - barcode assignmennt CHEQ-seq enhancer tiling B

description:
processed data file:
CHEQ-seq_Enhancer-tiling-B_enh-BC_list.tsv.gz


title: 8_ICRF_young


title: P2S_48h_29

description: single embryo for total RNA-seq

tag: batch

value: 2nd batch


title: 12_cPP_rep3

description: Central pulp rep3

tag: Sex


title: Enhancer - barcode assignmennt CHEQ-seq ATAC

description:
processed data file:
CHEQ-seq_ATAC_enh-BC_list.tsv.gz


title: Enhancer - barcode assignmennt CHEQ-seq enhancer tiling A

description:
processed data file:
CHEQ-seq_Enhancer-tiling-A_enh-BC_list.tsv.gz


title: Cerebral Cortex_Ctrl_Rep1

description: uninjured

value: Cerebral cortex

value: No


title: 9_ICRF_young


title: 5_ICRF_young


title: Al-NTL4-OX-rep3

city: Guang Zhou

first: Yan

last: Lin

state: Guang Dong

address: 广州市石榴岗路10号

department: Institute of Nanfan ＆Seed Industry

institute: Guangdong Academy of Sciences

data_processing:
Trim Smart-seq2 public primer sequence from the Reads (If the length of trimmed Reads is lower than 30bp, reads should be discarded. If it is PE sequence, both of the reads must be removed
Remove the contaminated reads for adapters (The contaminated reads for adapters was defined that the Read Bases contained more than 5bp of adapter sequences. If either one of the PE Reads was polluted, we will remove both ends of the reads)
Remove the low quality reads (The low quality reads was defined that the number of Reads Bases whose phred Quality value was less than or equal to 19 accounted for more than 15% . If either one of the PE Reads defined as low quality, we will remove both ends of the reads);
Remove the reads whose N base more than 5% for total bases (If either one of the PE Reads had high Ns bases, we will remove both ends of the reads);
Screening DEGs using NOISeq.
Supplementary_files_format_and_content: Reads Count for each gene in each sample was counted by HTSeq v0.6.0,FPKM (Fragments Per Kilobase Millon Mapped Reads) was then calculated to estimate the expression level of genes in each sample

organism: Arabidopsis thaliana

source_name: Root

extract_protocol:
Ethanol precipitation protocol and CTAB-PBIOZOL reagent for the purification of total RNA from the plant tissue
BGI RNA-Seq kit

tag: genotype/variation

tag: day after al


title: P1L_48h_8

description: single embryo for total RNA-seq

value: 1st batch


title: P2S_48h_18

description: single embryo for total RNA-seq


title: 9_pPP_rep3

description: Peripheral pulp rep3

source_name: Adult contour feather (peripheral pulp)

value: Peripheral pulp


title: 6_ICRF_young


title: Al-NTL4-OX-rep2


title: P1L_48h_4

description: single embryo for total RNA-seq


title: 10_cPP_rep1

description: Central pulp rep1

value: white leghorn


title: 8_pPP_rep2

description: Peripheral pulp rep2

value: male


title: Al-NTL4-OX-rep1


title: 4_ICRF_young


title: 3_ICRF_young


title: P1L_48h_6

description: single embryo for total RNA-seq


title: P2S_48h_30

description: single embryo for total RNA-seq


title: 7_pPP_rep1

description: Peripheral pulp rep1


title: Al-WT-rep3

value: WT


title: P1S_48h_15

description: single embryo for total RNA-seq


title: 6_DS_rep3

description: Dermal sheath rep3

source_name: Adult contour feather (dermal sheath)

value: Dermal sheath


title: 2_ICRF_young

value: 2-2.5 month old (young)

value: GV oocyte - SN


title: 5_DS_rep2

description: Dermal sheath rep2


title: 1_ICRF_young


title: P1S_48h_8

description: single embryo for total RNA-seq

value: worker embryo


title: Al-WT-rep1


title: Al-WT-rep2


title: P1S_48h_7

description: single embryo for total RNA-seq


title: CK-NTL4-OX-rep3


title: rbGFP rep2

city: Muenster

first: Hyun-Woo

last: Jeong

state: NRW

department: Dept. Tissue Morphogenesis

country: Germany

institute: Max-Planck-Institute for Molecular Biomedicine

data_processing:
illumina bcl2fastq (v2.19.1.403) used for basecalling and demultiplexing
UMI-tools (v1.0.0) used for cell barcode and UMI counting
STAR (v2.7.3a) used for mouse reference genome (mm10) alignment
Subread featureCounts (v1.6.4) used for UMI quantification
Genome_build: mm10
Supplementary_files_format_and_content: tsv files include UMI counts for each cell barcode and genes

source_name: PdgfrbCre GFP+ cells

extract_protocol:
3-week-old (n=2) male mice were used for non-hematopoietic single cell analysis. Femur and tibia were harvested, cleaned from attached surrounding tissue and collected in ice cold sterile PBS (without Ca2+/Mg2+). Next, bones were cut into small pieces and crushed using mortar and pestle. Samples were digested using 1 ml of collagenase type II and I enzymes (2mg/ml) for 20mins at 37°C under gentle agitation. Digested samples were transferred to 70μm strainers in 50ml tubes to obtain a single–cell suspension, which was resuspended in blocking solution (1% BSA, 1mM EDTA in PBS without Ca2+/Mg2+), centrifuged at 300g for 5mins, washed 2 to 3 times with ice-cold blocking solution, and filtered through 50μm strainers. Pellets were resuspended in the equivalent volume of blocking solution. To enrich bone marrow stromal cells, hematopoietic cells were removed using a lineage cell depletion kit (MACS, cat#130-090-858) following the manufacturer’s instructions. Next, lineage negative cells were depleted by CD45 and CD117 using microbeads (MACS, cat. no. 130-052-301 and 130-091-224) from lineage negative (lin-) bone cells. Bone marrow stromal cells were examined under microscopy and resuspended at 106 cells/ml in 0.05% BSA in PBS. For Pdgfrb-CreERT2 Rosa26-mT/mG reporter mice (n=4), bone single cell suspension was prepared and lineage-positive cells were depleted as described above. GFP positive cells were sorted by FACS Aria II (BD Bioscience), collected in blocking buffer and pused for scRNA sequencing.
Bone marrow stromal single cells or Pdgfrb-CreERT2 GFP+ sorted cells were prepared microfluidic droplets-based single cell RNA sequencing (10XGenomics). Single cells were encapsulated into emulsion droplets using Chromium controller (10X genomics). The scRNA seq libraries were prepared using the chromium single cells 3’ regent kit (V2) (10X genomics, cat#PN-120237) according to the manufacturer’s protocol. scRNA-seq libraries were evaluated and quantified by Agilent Bioanalyzer using High sensitivity DNA kit (cat#5067-4626) and Qubit (ThermoFisher, car#Q32851). Individual libraries were diluted to 4nM and pooled for sequencing. Pooled libraries were sequenced by High Output kit (150 cycle) (Illumina cat#20024907) with Next500/550 sequencer (Illumina).

value: C57BL/6 :PdgfrbCreERT2; R26 mTmG

value: Bone

tag: cell type

value: PdgfrbCre GFP+ cells

value: postnatal day 21


title: 4_DS_rep1

description: Dermal sheath rep1


title: P2L_24h_8

description: single embryo for total RNA-seq

value: drone embryo

value: embryonic development 24h


title: CK-NTL4-OX-rep2


title: rbGFP rep1


title: 2_DP_rep2

description: Dermal papilla rep2

source_name: Adult contour feather (dermal papilla)

value: Dermal papilla


title: BSC rep4

source_name: Bone stromal cells

value: Bone stromal cells


title: CK-NTL4-OX-rep1


title: BSC rep3


title: 1_DP_rep1

description: Dermal papilla rep1


title: CK-WT-rep3


title: P2S_24h_32

description: single embryo for total RNA-seq

value: 2nd batch


title: BSC rep2


title: P2L_24h_11

description: single embryo for total RNA-seq


title: con_2

description:
expressed_gene_FPKM.txt
expressed_gene_reads.txt

data_processing:
Illumina bcl2fastq software used for basecalling.
3' adapter (end1:AGATCGGAAGAGC;end2:AGATCGGAAGAGC) and low quality bases trimmed using FASTX-Toolkit (Version 0.0.13). the short reads less than 16nt were also dropped.
Reads were mapped using tophat2 --read-edit-dist 4 -N 4 --b2-N 1 -p 16 --no-discordant. Uniq mapped reads were remained. while several mapped reads have the same start position and end position, only one is considered.
edgeR was used to the DEG analysis with FC>2 and Qvalue <= 0.05
Genome_build: GRCm38
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample.

source_name: Kindey with control treatment

growth_protocol: The mice were housed in a room with a suitable temperature and a relative humidity of 50%–60%. Eight-week-old male mouse was used for this study after 1 week of adaption.

extract_protocol:
For the RNA-seq library, the total RNA was extracted from renal tissue samples by using TRIzol Reagent (Ambion) following the manufacturer’s  instructions. The total RNA was treated with RQ1 DNase (Promega) to remove DNA. The quality and quantity of the purified RNA were determined by measuring the  absorbance at 260 nm/280 nm (A260/A280) using Smartspec Plus (BioRad). The RNA integrity was further verified by 1.5% agarose gel electrophoresis. For each sample, 5 μg of the total RNA was used to prepare the RNA-seq library. The Ribosomal RNAs were depleted with rRNA (Human/Mouse/Rat) Removal Kit  (Ribo-Zero™, RZH1046, Epicentre, and MGIEasy, 1000005953 for the tissue and HK-2 cell samples, respectively) before they were used for directional RNA-seq library preparation.
Purified mRNAs were iron-fragmented at 95 °C, followed by end repair and 5′ adaptor ligation. Then, reverse transcription was performed with RT primer harboring the 3′ adaptor sequence and randomized hexamer. The cDNAs were purified and PCR amplified. PCR products corresponding to 200–500 bps was purified, quantified, and stored at −80 °C until they were used for sequencing. For high-throughput sequencing, the libraries were prepared following the manufacturer’s instructions and applied to Illumina HiSeq X Ten system for 150 nt paired-end sequencing.

treatment_protocol: Eighteen mice were randomly divided into three groups and injected with either physiological saline (control) or cisplatin (Sigma, St. Louis, MO, USA) at a dose of 30 mg/kg body weight for 24 h or 48 h before killing the animal.

value: kindey

value: injected with physiological saline


title: CK-WT-rep2


title: P2S_24h_34

description: single embryo for total RNA-seq


title: con_1


title: BSC rep1

value: male


title: P2S_24h_35

description: single embryo for total RNA-seq


title: CIS_2

source_name: Kindey with cisplatin treatment

value: injected with cisplatin


title: dpMSC-3

data_processing:
Illumina RTA v2 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm10 whole genome using TopHat-2 v2.1.1 with parameters -g 2
HTSeq-count was used to count the aligned reads on a per gene basis (Version: HTSeq-0.11.2). The count data were normalized, fragments per kilobase of transcript per million of sequencing reads (FPKM) values were calculated using the DESeq2 package
Genome_build: mm10
Supplementary_files_format_and_content: txt file includes FPKM values for each gene

source_name: diaphysis mesenchymal stromal cells

extract_protocol:
Femur and tibia were harvested from 3-week-old mice, surrounding muscle tissue was cleaned off and the epiphysis removed. The metaphysis was separated with a scalpel to obtain metaphyseal derived MSC (mp-MSCs). Next, bone marrow was gently removed from the bone shaft into suspension medium (1% FBS, 1mM EDTA in PBS without Ca2+/Mg2+) to obtain BM-derived MSCs (bm-MSCs). Metaphyseal fragments were cut into small pieces and crushed using mortar and pestle. Crushed samples were digested using 1 ml of Collagenase type II and I enzymes (2mg/ml) for 20mins at 37°C under gentle agitation. Digested metaphysis and BM samples were transferred through 70μm strainers into 50ml tubes to obtain single–cell suspensions, which were resuspended in suspension medium, centrifuged at 300g for 5mins, and washed 2 to 3 times. Cell pellets were resuspended in the respective volume of FACS buffer. Single cell suspensions were prepared from 3-weeks-old Hey1-GFP, Pdgfra-H2B-GFP reporter mice. Cell sorting was performed on a FACS Aria II cell sorter (BD bioscience). Dead cells and debris were excluded by FSC, SSC and DAPI positive signal. Sorted GFP were collected in  RTL buffer for bulk RNA-seq.
RNA was isolated using the RNeasy Plus Micro Kit (QIAGEN) according to the manufacturer’s instructions. RNA quality was checked using a 2100 BioAnalyzer (Agilent). 10ng of RNA were used for preparation of sequencing libraries with SMARTer Ultra Low input RNA Kit-V3 (Clontech laboratories) and followed by Nextera XT DNA Library Prep Kit (Illumina) according to the manufacturer’s instructions. Libraries were validated using a BioAnalyzer, quantified by qPCR and Qubit Fluorometric Quantitation (Thermo Fisher). Libraries were diluted to a final concentration of 1.6pM for sequencing. The NextSeq 500/550 Mid Output Kit v2.5 (Illumina) was used for sequencing with a NextSeq 500 (Illumina).

value: C57BL/6 :Pdgfra-H2B GFP

value: diaphysis mesenchymal stromal cells


title: P1L_24h_5

description: single embryo for total RNA-seq

source_name: embryo

value: 1st batch


title: CIS_1


title: Ron1-KO-rep3

city: jinan

first: Jizheng

last: song

address: guyunhu

department: school of bioengineering

institute: qilu university of technology

data_processing:
Sequencing of RNA samples was performed on the BGISEQ-500 platform at BGI-Shenzhen, China.
Entire clean reads were mapped to the reference genome with HISAT2 (v2.0.4)
Bowtie2 (v2.2.5) was applied to align the clean reads to the reference coding gene set, and then the gene expression level was evaluated by RSEM (v1.2.12)
Genome_build: GCA_000280675.1
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

organism: Beauveria bassiana ARSEF 2860

source_name: Beauveria bassiana ARSEF 2860

growth_protocol: WT and Ron1：culture on chitin medium

extract_protocol:
Total RNA was extracted from fungal cultures of each mode with TRIzol-A+ reagent.
RNA libraries were prepared for sequencing using standard BGISEQ-500 protocols

treatment_protocol: Disruption Ron1 by homologous recombination

value: ARSEF 2860

value: Ron1 KO


title: dpMSC-2

value: Bone


title: 3_DP_rep3

description: Dermal papilla rep3

value: white leghorn


title: P2L_24h_9

description: single embryo for total RNA-seq


title: Ron1-KO-rep1


title: Ron1-KO-rep2


title: WT-rep3

value: WT


title: P1L_24h_19

description: single embryo for total RNA-seq

value: drone embryo


title: P1L_24h_26

description: single embryo for total RNA-seq

value: embryonic development 24h


title: M4

city: Bodø

first: Ioannis

last: Konstantinidis

state: Nordland

address: Universitetsalléen 11

department: Faculty of Biosciences and Aquaculture

country: Norway

institute: Nord University

data_processing:
RNA-seq reads were decontaminated by BBDuk (v. 37.61) using the following parameters ktrim=r k=23 mink=11 hdist=1 tpe tbo qtrim=r trimq=30 minlen=23
Reads were aligned to the genome by HISAT2 (v. 2.1.0) using the core hisat2 function with default parameters
Reads were counted by feature Counts of the Subread package
Genome_build: GCA_001858045.3
Supplementary_files_format_and_content: A plain text file is provided with the feature counts per gene per sample

organism: Oreochromis niloticus

source_name: Fast muscle

extract_protocol:
Fast muscle was stored in -80 C. RNA was extracted after homogenizing the tissue within the DNA/RNA Shield using the Quick-RNA miniprep plus kit (Zymo Research) according to manufacturer's protocol and recommendations.
After extracting the total RNA as described above, rRNA depletion was performed using the Ribo-Zero Gold rRNA removal kit (Illumina) according to manufacturer's protocol
RNA libraries were prepared using the NEBNext Ultra II directional RNA library prep kit for Illumina a ccording to manufacturer's protocol

tag: replicates

value: replicate 4

value: 5 months

value: Fast (white) muscle


title: mpMSC-4

source_name: Metaphyseal mesenchymal stromal cells

tag: strain

value: C57BL/6 :Hey1-GFP

value: Metaphyseal mesenchymal stromal cells

value: postnatal day 21


title: M5

tag: gender

value: male

value: replicate 5


title: P1S_24h_9

description: single embryo for total RNA-seq

value: worker embryo


title: WT-rep2


title: M3

value: replicate 3


title: P1S_24h_15

description: single embryo for total RNA-seq

value: 1st batch


title: mpMSC-2


title: mpMSC-3


title: CK-WT-rep1


title: M2

value: replicate 2


title: WT-rep1

value: ARSEF 2860


title: P1S_24h_6

description: single embryo for total RNA-seq


title: mpMSC-1

value: Bone


title: dpMSC-1

value: C57BL/6 :Pdgfra-H2B GFP

value: diaphysis mesenchymal stromal cells


title: MNaseseq_Input_yFR1206_WT_R2

status: Public on Jul 26 2021

city: Montreal

first: Francois

last: Robert

state: QC

address: 110 av des Pins Ouest

country: Canada

institute: IRCM

description:
Barcode ID: UDI0049 from Illumina TruSeq Kits
Biological replicate 2 of 2. Input signal of WT cells. This Input DNA was obtained from the same yeast extract used to perform H4 MNase-ChIP-seq.

data_processing:
Paired-end reads from MNase-ChIP-seq data where aligned on the S. cerevisiae genome version sacCer3 using Bowtie2 version 2.3.4.3 (Langmead and Salzberg, 2012) with the parameter “-X 1000” to include longer fragments. Unmapped and low-quality fragments were removed using SAMtools version 1.9 using the following parameters “view -f 2 -F 2048”. Duplicates were removed using SAMtools with the following parameter “fixmate -m” and then “markdup -r”. The aligned files were converted to BED using BEDTools version 2.27.1 using a custom software called seqtools version 1.0 available at https://github.com/francoisrobertlab/seqtools with the command “seqtools bam2bed” resulting in BED files containing the DNA fragments. The BED files of replicates were merged using seqtools with command “seqtools merge”. Genome coverage files were generated using customs scripts “seqtools” with commands “mnasetools prepgenomecov” and “seqtools genomecov” that require BEDTools version 2.27.1 and KentUtils (downloaded on July 16th 2018). The genome coverage files were converted to the bigWig format using the utilities from UCSC Genome Browser (Casper et al., 2018).
Genome_build: S. cerevisiae (UCSC sacCer3)
Supplementary_files_format_and_content: bigWig files for both individual replicates (beginning with "CJ") and the combined biological replicates (beginning with "MNase").

source_name: Yeast cell extract

growth_protocol: Cells were grown at 30°C and 200 rpm in YPD (yeast extract-peptone-2% glucose, supplemented with 44 µM adenine) medium as follow. Strains were streaked from glycerol stocks onto 2% agar YPD plates and grown at 30°C for 2-3 days. An isolated colony was then grown overnight in 20 mL of YPD. This preculture was used to inoculate 500 mL of YPD at an OD600 of 0.1 which was grown to an OD600 of 0.7-0.9.

extract_protocol: MNase-ChIP experiments were performed in two biological replicates, as previously described (Rando, 2010) with minor modifications. Briefly, yeast cultures were grown in 500 mL of YPD to an OD600 of 0.7-0.9 before crosslinking with 1% formaldehyde (Fisher Scientific, BP531-500) at room temperature for 15 min and quenched with 125 mM glycine at room temperature for 5 min. Crosslinked cells were collected by centrifugation and washed twice with ice-cold ddH2O. Yeast cell wall digestion was then performed by resuspending cell pellets in 39 mL Buffer Z (1 M sorbitol and 50 mM Tris-HCl pH 7.4) containing 10 mM β-mercaptoethanol and 10 mg Zymolyase 100T (US Biological, Z1004) and incubating at 30˚C with agitation (20-40 min). When digestion was completed, the resulting spheroplasts were pelleted by centrifugation at 3,000 g for 5 min at 4°C and gently resuspended in 2.5 mL of NP buffer (1 M sorbitol, 50 mM NaCl, 10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 µM spermidine (Sigma-Aldrich, 85558), 1 mM β-mercaptoethanol and 0.075% NP-40) before being exposed to MNase (Worthington, LS004798) digestion titration to generate the appropriate size of chromatin fragments as follow. 700 µl of spheroplasts were incubated with 4.5, 9, 18, and 36 units of MNase (resuspended in 10 mM Tris-HCl pH 7.4 and 30% glycerol) at 37˚C for 20 min and reaction was stopped by adding 10 mM EDTA. To ensure IP conditions, 200 µL of adjusting buffer (50 mM HEPES-KOH pH7.5, 140 mM NaCl, 1% Triton X-100, and 0.1% Na-deoxycholate) were added to the digested chromatin sample. To determine the appropriate condition of MNase digestion, 45 µL (5%) of each MNase digested sample were taken and incubated with 5 μL of 10X TE/SDS at 65˚C for 16 hr to reverse crosslinking. Samples were then treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 100 % ethanol. Precipitated DNA was resuspended in 30 μL of TE, treated with 1µL of 10 mg/mL RNAse A at 37°C for 1 hr before being analyzed by electrophoresis on a 1.8% TAE-agarose gel stained with ethidium bromide. The digested samples were also qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA Kit (Agilent Technologies, 5067-4626). The condition giving approximately 65% mono-, 25% di- and 10% tri-nucleosomes was subjected to IP as follow. 45µL (5%) were saved as Input sample and the remaining digested chromatin was incubated overnight with the appropriate antibody-coupled Dynabeads as per our standard ChIP protocol (see above). Beads were then washed and samples reverse crosslinked, treated with RNase A, and subsequently subjected to Proteinase K before being twice phenol/chloroform/isoamyl alcohol extracted followed by precipitation with NaCl and ethanol. Precipitated DNA was resuspended in 50 μL of TE before being used in sequencing libraries.  Before proceeding with MNase-ChIP-seq libraries, ChIP and Input samples were subjected to a 2X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions. Samples were eluted with 50 μL of Elution buffer (10 mM Tris pH 8.0) and quantified/qualified on an Agilent 2100 Bioanalyzer instrument using the High Sensitivity DNA kit.  1 ng of Input DNA and 40 μL of ChIP DNA were used for library preparation as follow. The ends of DNA were repaired by incubating in 70 μL of 1X NEBuffer 2 containing 0.6 units of T4 DNA polymerase (NEB, M0203L), 2 units of T4 polynucleotide kinase (NEB, M0201S), 0.09 nM dNTPs and 0.045 μg/μL of BSA at 12˚C for 30 min. Repaired DNA was then subjected to a 2X cleanup using KAPA Pure Beads before dA-tailing as follow. Beads containing the repaired DNA were resuspended in 50 μL of 1X NEBuffer 2 containing 0.1 mM dATP and 25 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M), and incubated at 37˚C for 30 min. After a 2X cleanup with KAPA Pure Beads, dA-tailed DNA was ligated to index adapters (Roche, SeqCap Adapter kit A (07141530001) and SeqCap Adapter kit B (07141548001)) as follow. The beads were resuspended in 45 μL of 1X Ligase buffer containing 8 nM of adapter and 2.5 units of T4 DNA ligase and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1X cleanup with KAPA Pure Beads. Libraries were PCR-amplified with 10-12 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and cleaned up using 1X KAPA Pure Beads. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library was pooled (12 libraries per pool) and subjected to sequencing on Illumina HiSeq 4000 or NovaSeq 6000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.

treatment_protocol: Cultures were crosslinked with 1% formaldehyde (Fisher Scientific, BP531-500) at room temperature for 30 min. Crosslinking was quenched with 125 mM glycine.

tag: strain name

tag: chip antibody


title: MNase-input_Pob3_yFR930chd1D_R2

description:
Biological replicate 2 of 2. Input signal of chd1D cells. This Input DNA was obtained from the same yeast extract used to perform Pob3 MNase-ChIP-seq.
processed data file (two biological replicates combined):  MNaseInput_Pob3_yFR930chd1D_CJ7-CJ8comb_20200319.bw

extract_protocol:
MNase-ChIP experiments were performed in two biological replicates, as previously described (Rando, 2010) with minor modifications. Briefly, yeast cultures were grown in 500 mL of YPD to an OD600 of 0.7-0.9 before crosslinking with 1% formaldehyde (Fisher Scientific, BP531-500) at room temperature for 15 min and quenched with 125 mM glycine at room temperature for 5 min. Crosslinked cells were collected by centrifugation and washed twice with ice-cold ddH2O. Yeast cell wall digestion was then performed by resuspending cell pellets in 39 mL Buffer Z (1 M sorbitol and 50 mM Tris-HCl pH 7.4) containing 10 mM β-mercaptoethanol and 10 mg Zymolyase 100T (US Biological, Z1004) and incubating at 30˚C with agitation (20-40 min). When digestion was completed, the resulting spheroplasts were pelleted by centrifugation at 3,000 g for 5 min at 4°C and gently resuspended in 2.5 mL of NP buffer (1 M sorbitol, 50 mM NaCl, 10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 µM spermidine (Sigma-Aldrich, 85558), 1 mM β-mercaptoethanol and 0.075% NP-40) before being exposed to MNase (Worthington, LS004798) digestion titration to generate the appropriate size of chromatin fragments as follow. 700 µl of spheroplasts were incubated with 4.5, 9, 18, and 36 units of MNase (resuspended in 10 mM Tris-HCl pH 7.4 and 30% glycerol) at 37˚C for 20 min and reaction was stopped by adding 10 mM EDTA. To ensure IP conditions, 200 µL of adjusting buffer (50 mM HEPES-KOH pH7.5, 140 mM NaCl, 1% Triton X-100, and 0.1% Na-deoxycholate) were added to the digested chromatin sample. To determine the appropriate condition of MNase digestion, 45 µL (5%) of each MNase digested sample were taken and incubated with 5 μL of 10X TE/SDS at 65˚C for 16 hr to reverse crosslinking. Samples were then treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 100 % ethanol. Precipitated DNA was resuspended in 30 μL of TE, treated with 1µL of 10 mg/mL RNAse A at 37°C for 1 hr before being analyzed by electrophoresis on a 1.8% TAE-agarose gel stained with ethidium bromide. The digested samples were also qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA Kit (Agilent Technologies, 5067-4626). The condition giving approximately 65% mono-, 25% di- and 10% tri-nucleosomes was subjected to IP as follow. 45µL (5%) were saved as Input sample and the remaining digested chromatin was incubated overnight with the appropriate antibody-coupled Dynabeads as per our standard ChIP protocol (see above). Beads were then washed and samples reverse crosslinked, treated with RNase A, and subsequently subjected to Proteinase K before being twice phenol/chloroform/isoamyl alcohol extracted followed by precipitation with NaCl and ethanol. Precipitated DNA was resuspended in 50 μL of TE before being used in sequencing libraries.  Before proceeding with MNase-ChIP-seq libraries, ChIP and Input samples were subjected to a 2X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions. Samples were eluted with 50 μL of Elution buffer (10 mM Tris pH 8.0) and quantified/qualified on an Agilent 2100 Bioanalyzer instrument using the High Sensitivity DNA kit.  1 ng of Input DNA and 40 μL of ChIP DNA were used for library preparation as follow. The ends of DNA were repaired by incubating in 70 μL of 1X NEBuffer 2 containing 0.6 units of T4 DNA polymerase (NEB, M0203L), 2 units of T4 polynucleotide kinase (NEB, M0201S), 0.09 nM dNTPs and 0.045 μg/μL of BSA at 12˚C for 30 min. Repaired DNA was then subjected to a 2X cleanup using KAPA Pure Beads before dA-tailing as follow. Beads containing the repaired DNA were resuspended in 50 μL of 1X NEBuffer 2 containing 0.1 mM dATP and 25 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M), and incubated at 37˚C for 30 min. After a 2X cleanup with KAPA Pure Beads, dA-tailed DNA was ligated to index adapters (Roche, SeqCap Adapter kit A (07141530001) and SeqCap Adapter kit B (07141548001)) as follow. The beads were resuspended in 45 μL of 1X Ligase buffer containing 8 nM of adapter and 2.5 units of T4 DNA ligase and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1X cleanup with KAPA Pure Beads. Libraries were PCR-amplified with 10-12 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and cleaned up using 1X KAPA Pure Beads. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library was pooled (12 libraries per pool) and subjected to sequencing on Illumina HiSeq 4000 or NovaSeq 6000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID:Adapter 25 from SeqCap Adapter kits A and B (Roche, 07141530001 and 07141548001)
MNase-ChIP-Seq

tag: genotype


title: MNaseseq_Input_yFR1206_WT_R1

description:
Barcode ID: UDI0048 from Illumina TruSeq Kits
Biological replicate 1 of 2. Input signal of WT cells. This Input DNA was obtained from the same yeast extract used to perform H4 MNase-ChIP-seq.
processed data file (two biological replicates combined): MNaseseq_Input_yFR1206_WT_CJ15-CJ16_20201127-cov-1.1.bw


title: MNaseseq_IP_H4_yFR1206_WT_R2

description:
Barcode ID: UDI0033 from Illumina TruSeq Kits
Biological replicate 2 of 2. H4 occupancy by MNase-ChIP-seq in WT cells. H4 MNase-ChIP-seq was performed using a rabbit polyclonal antibody against H4.

value: anti-H4 (a gift from A. Verreault, 2 uL)


title: MNase-input_Pob3_yFR116wt_R1

description:
Biological replicate 1 of 2. Input signal of WT cells. This Input DNA was obtained from the same yeast extract used to perform Pob3 MNase-ChIP-seq.
processed data file (two biological replicates combined):  MNaseInput_Pob3_yFR116WT_CJ5-CJ6comb_20200319.bw

extract_protocol:
MNase-ChIP experiments were performed in two biological replicates, as previously described (Rando, 2010) with minor modifications. Briefly, yeast cultures were grown in 500 mL of YPD to an OD600 of 0.7-0.9 before crosslinking with 1% formaldehyde (Fisher Scientific, BP531-500) at room temperature for 15 min and quenched with 125 mM glycine at room temperature for 5 min. Crosslinked cells were collected by centrifugation and washed twice with ice-cold ddH2O. Yeast cell wall digestion was then performed by resuspending cell pellets in 39 mL Buffer Z (1 M sorbitol and 50 mM Tris-HCl pH 7.4) containing 10 mM β-mercaptoethanol and 10 mg Zymolyase 100T (US Biological, Z1004) and incubating at 30˚C with agitation (20-40 min). When digestion was completed, the resulting spheroplasts were pelleted by centrifugation at 3,000 g for 5 min at 4°C and gently resuspended in 2.5 mL of NP buffer (1 M sorbitol, 50 mM NaCl, 10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 µM spermidine (Sigma-Aldrich, 85558), 1 mM β-mercaptoethanol and 0.075% NP-40) before being exposed to MNase (Worthington, LS004798) digestion titration to generate the appropriate size of chromatin fragments as follow. 700 µl of spheroplasts were incubated with 4.5, 9, 18, and 36 units of MNase (resuspended in 10 mM Tris-HCl pH 7.4 and 30% glycerol) at 37˚C for 20 min and reaction was stopped by adding 10 mM EDTA. To ensure IP conditions, 200 µL of adjusting buffer (50 mM HEPES-KOH pH7.5, 140 mM NaCl, 1% Triton X-100, and 0.1% Na-deoxycholate) were added to the digested chromatin sample. To determine the appropriate condition of MNase digestion, 45 µL (5%) of each MNase digested sample were taken and incubated with 5 μL of 10X TE/SDS at 65˚C for 16 hr to reverse crosslinking. Samples were then treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 100 % ethanol. Precipitated DNA was resuspended in 30 μL of TE, treated with 1µL of 10 mg/mL RNAse A at 37°C for 1 hr before being analyzed by electrophoresis on a 1.8% TAE-agarose gel stained with ethidium bromide. The digested samples were also qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA Kit (Agilent Technologies, 5067-4626). The condition giving approximately 65% mono-, 25% di- and 10% tri-nucleosomes was subjected to IP as follow. 45µL (5%) were saved as Input sample and the remaining digested chromatin was incubated overnight with the appropriate antibody-coupled Dynabeads as per our standard ChIP protocol (see above). Beads were then washed and samples reverse crosslinked, treated with RNase A, and subsequently subjected to Proteinase K before being twice phenol/chloroform/isoamyl alcohol extracted followed by precipitation with NaCl and ethanol. Precipitated DNA was resuspended in 50 μL of TE before being used in sequencing libraries.  Before proceeding with MNase-ChIP-seq libraries, ChIP and Input samples were subjected to a 2X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions. Samples were eluted with 50 μL of Elution buffer (10 mM Tris pH 8.0) and quantified/qualified on an Agilent 2100 Bioanalyzer instrument using the High Sensitivity DNA kit.  1 ng of Input DNA and 40 μL of ChIP DNA were used for library preparation as follow. The ends of DNA were repaired by incubating in 70 μL of 1X NEBuffer 2 containing 0.6 units of T4 DNA polymerase (NEB, M0203L), 2 units of T4 polynucleotide kinase (NEB, M0201S), 0.09 nM dNTPs and 0.045 μg/μL of BSA at 12˚C for 30 min. Repaired DNA was then subjected to a 2X cleanup using KAPA Pure Beads before dA-tailing as follow. Beads containing the repaired DNA were resuspended in 50 μL of 1X NEBuffer 2 containing 0.1 mM dATP and 25 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M), and incubated at 37˚C for 30 min. After a 2X cleanup with KAPA Pure Beads, dA-tailed DNA was ligated to index adapters (Roche, SeqCap Adapter kit A (07141530001) and SeqCap Adapter kit B (07141548001)) as follow. The beads were resuspended in 45 μL of 1X Ligase buffer containing 8 nM of adapter and 2.5 units of T4 DNA ligase and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1X cleanup with KAPA Pure Beads. Libraries were PCR-amplified with 10-12 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and cleaned up using 1X KAPA Pure Beads. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library was pooled (12 libraries per pool) and subjected to sequencing on Illumina HiSeq 4000 or NovaSeq 6000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID:Adapter 20 from SeqCap Adapter kits A and B (Roche, 07141530001 and 07141548001)
MNase-ChIP-Seq


title: MNase-input_Pob3_yFR930chd1D_R1

description:
Biological replicate 1 of 2. Input signal of chd1D cells. This Input DNA was obtained from the same yeast extract used to perform Pob3 MNase-ChIP-seq.
processed data file (two biological replicates combined):  MNaseInput_Pob3_yFR930chd1D_CJ7-CJ8comb_20200319.bw

extract_protocol:
MNase-ChIP experiments were performed in two biological replicates, as previously described (Rando, 2010) with minor modifications. Briefly, yeast cultures were grown in 500 mL of YPD to an OD600 of 0.7-0.9 before crosslinking with 1% formaldehyde (Fisher Scientific, BP531-500) at room temperature for 15 min and quenched with 125 mM glycine at room temperature for 5 min. Crosslinked cells were collected by centrifugation and washed twice with ice-cold ddH2O. Yeast cell wall digestion was then performed by resuspending cell pellets in 39 mL Buffer Z (1 M sorbitol and 50 mM Tris-HCl pH 7.4) containing 10 mM β-mercaptoethanol and 10 mg Zymolyase 100T (US Biological, Z1004) and incubating at 30˚C with agitation (20-40 min). When digestion was completed, the resulting spheroplasts were pelleted by centrifugation at 3,000 g for 5 min at 4°C and gently resuspended in 2.5 mL of NP buffer (1 M sorbitol, 50 mM NaCl, 10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 µM spermidine (Sigma-Aldrich, 85558), 1 mM β-mercaptoethanol and 0.075% NP-40) before being exposed to MNase (Worthington, LS004798) digestion titration to generate the appropriate size of chromatin fragments as follow. 700 µl of spheroplasts were incubated with 4.5, 9, 18, and 36 units of MNase (resuspended in 10 mM Tris-HCl pH 7.4 and 30% glycerol) at 37˚C for 20 min and reaction was stopped by adding 10 mM EDTA. To ensure IP conditions, 200 µL of adjusting buffer (50 mM HEPES-KOH pH7.5, 140 mM NaCl, 1% Triton X-100, and 0.1% Na-deoxycholate) were added to the digested chromatin sample. To determine the appropriate condition of MNase digestion, 45 µL (5%) of each MNase digested sample were taken and incubated with 5 μL of 10X TE/SDS at 65˚C for 16 hr to reverse crosslinking. Samples were then treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 100 % ethanol. Precipitated DNA was resuspended in 30 μL of TE, treated with 1µL of 10 mg/mL RNAse A at 37°C for 1 hr before being analyzed by electrophoresis on a 1.8% TAE-agarose gel stained with ethidium bromide. The digested samples were also qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA Kit (Agilent Technologies, 5067-4626). The condition giving approximately 65% mono-, 25% di- and 10% tri-nucleosomes was subjected to IP as follow. 45µL (5%) were saved as Input sample and the remaining digested chromatin was incubated overnight with the appropriate antibody-coupled Dynabeads as per our standard ChIP protocol (see above). Beads were then washed and samples reverse crosslinked, treated with RNase A, and subsequently subjected to Proteinase K before being twice phenol/chloroform/isoamyl alcohol extracted followed by precipitation with NaCl and ethanol. Precipitated DNA was resuspended in 50 μL of TE before being used in sequencing libraries.  Before proceeding with MNase-ChIP-seq libraries, ChIP and Input samples were subjected to a 2X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions. Samples were eluted with 50 μL of Elution buffer (10 mM Tris pH 8.0) and quantified/qualified on an Agilent 2100 Bioanalyzer instrument using the High Sensitivity DNA kit.  1 ng of Input DNA and 40 μL of ChIP DNA were used for library preparation as follow. The ends of DNA were repaired by incubating in 70 μL of 1X NEBuffer 2 containing 0.6 units of T4 DNA polymerase (NEB, M0203L), 2 units of T4 polynucleotide kinase (NEB, M0201S), 0.09 nM dNTPs and 0.045 μg/μL of BSA at 12˚C for 30 min. Repaired DNA was then subjected to a 2X cleanup using KAPA Pure Beads before dA-tailing as follow. Beads containing the repaired DNA were resuspended in 50 μL of 1X NEBuffer 2 containing 0.1 mM dATP and 25 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M), and incubated at 37˚C for 30 min. After a 2X cleanup with KAPA Pure Beads, dA-tailed DNA was ligated to index adapters (Roche, SeqCap Adapter kit A (07141530001) and SeqCap Adapter kit B (07141548001)) as follow. The beads were resuspended in 45 μL of 1X Ligase buffer containing 8 nM of adapter and 2.5 units of T4 DNA ligase and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1X cleanup with KAPA Pure Beads. Libraries were PCR-amplified with 10-12 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and cleaned up using 1X KAPA Pure Beads. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library was pooled (12 libraries per pool) and subjected to sequencing on Illumina HiSeq 4000 or NovaSeq 6000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID:Adapter 23 from SeqCap Adapter kits A and B (Roche, 07141530001 and 07141548001)
MNase-ChIP-Seq


title: MNase-ChIP_Pob3_yFR930chd1D_R2

description:
Biological replicate 2 of 2. Pob3 occupancy by MNase-ChIP-seq in chd1D cells. Pob3 MNase-ChIP-seq was performed using a rabbit polyclonal antibody against Pob3.
processed data file (two biological replicates combined):  MNaseChIP_Pob3_yFR930chd1D_CJ3-CJ4comb_20200319.bw

extract_protocol:
MNase-ChIP experiments were performed in two biological replicates, as previously described (Rando, 2010) with minor modifications. Briefly, yeast cultures were grown in 500 mL of YPD to an OD600 of 0.7-0.9 before crosslinking with 1% formaldehyde (Fisher Scientific, BP531-500) at room temperature for 15 min and quenched with 125 mM glycine at room temperature for 5 min. Crosslinked cells were collected by centrifugation and washed twice with ice-cold ddH2O. Yeast cell wall digestion was then performed by resuspending cell pellets in 39 mL Buffer Z (1 M sorbitol and 50 mM Tris-HCl pH 7.4) containing 10 mM β-mercaptoethanol and 10 mg Zymolyase 100T (US Biological, Z1004) and incubating at 30˚C with agitation (20-40 min). When digestion was completed, the resulting spheroplasts were pelleted by centrifugation at 3,000 g for 5 min at 4°C and gently resuspended in 2.5 mL of NP buffer (1 M sorbitol, 50 mM NaCl, 10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 µM spermidine (Sigma-Aldrich, 85558), 1 mM β-mercaptoethanol and 0.075% NP-40) before being exposed to MNase (Worthington, LS004798) digestion titration to generate the appropriate size of chromatin fragments as follow. 700 µl of spheroplasts were incubated with 4.5, 9, 18, and 36 units of MNase (resuspended in 10 mM Tris-HCl pH 7.4 and 30% glycerol) at 37˚C for 20 min and reaction was stopped by adding 10 mM EDTA. To ensure IP conditions, 200 µL of adjusting buffer (50 mM HEPES-KOH pH7.5, 140 mM NaCl, 1% Triton X-100, and 0.1% Na-deoxycholate) were added to the digested chromatin sample. To determine the appropriate condition of MNase digestion, 45 µL (5%) of each MNase digested sample were taken and incubated with 5 μL of 10X TE/SDS at 65˚C for 16 hr to reverse crosslinking. Samples were then treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 100 % ethanol. Precipitated DNA was resuspended in 30 μL of TE, treated with 1µL of 10 mg/mL RNAse A at 37°C for 1 hr before being analyzed by electrophoresis on a 1.8% TAE-agarose gel stained with ethidium bromide. The digested samples were also qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA Kit (Agilent Technologies, 5067-4626). The condition giving approximately 65% mono-, 25% di- and 10% tri-nucleosomes was subjected to IP as follow. 45µL (5%) were saved as Input sample and the remaining digested chromatin was incubated overnight with the appropriate antibody-coupled Dynabeads as per our standard ChIP protocol (see above). Beads were then washed and samples reverse crosslinked, treated with RNase A, and subsequently subjected to Proteinase K before being twice phenol/chloroform/isoamyl alcohol extracted followed by precipitation with NaCl and ethanol. Precipitated DNA was resuspended in 50 μL of TE before being used in sequencing libraries.  Before proceeding with MNase-ChIP-seq libraries, ChIP and Input samples were subjected to a 2X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions. Samples were eluted with 50 μL of Elution buffer (10 mM Tris pH 8.0) and quantified/qualified on an Agilent 2100 Bioanalyzer instrument using the High Sensitivity DNA kit.  1 ng of Input DNA and 40 μL of ChIP DNA were used for library preparation as follow. The ends of DNA were repaired by incubating in 70 μL of 1X NEBuffer 2 containing 0.6 units of T4 DNA polymerase (NEB, M0203L), 2 units of T4 polynucleotide kinase (NEB, M0201S), 0.09 nM dNTPs and 0.045 μg/μL of BSA at 12˚C for 30 min. Repaired DNA was then subjected to a 2X cleanup using KAPA Pure Beads before dA-tailing as follow. Beads containing the repaired DNA were resuspended in 50 μL of 1X NEBuffer 2 containing 0.1 mM dATP and 25 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M), and incubated at 37˚C for 30 min. After a 2X cleanup with KAPA Pure Beads, dA-tailed DNA was ligated to index adapters (Roche, SeqCap Adapter kit A (07141530001) and SeqCap Adapter kit B (07141548001)) as follow. The beads were resuspended in 45 μL of 1X Ligase buffer containing 8 nM of adapter and 2.5 units of T4 DNA ligase and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1X cleanup with KAPA Pure Beads. Libraries were PCR-amplified with 10-12 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and cleaned up using 1X KAPA Pure Beads. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library was pooled (12 libraries per pool) and subjected to sequencing on Illumina HiSeq 4000 or NovaSeq 6000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID:Adapter 18 from SeqCap Adapter kits A and B (Roche, 07141530001 and 07141548001)
MNase-ChIP-Seq

value: anti-Pob3 (a gift from T. Formosa, 1 μL)


title: MNase-ChIP_Pob3_yFR116wt_R2

description:
Biological replicate 2 of 2. Pob3 occupancy by MNase-ChIP-seq in WT cells. Pob3 MNase-ChIP-seq was performed using a rabbit polyclonal antibody against Pob3.
processed data file (two biological replicates combined):  MNaseChIP_Pob3_yFR116WT_CJ1-CJ2comb_20200319.bw

extract_protocol:
MNase-ChIP experiments were performed in two biological replicates, as previously described (Rando, 2010) with minor modifications. Briefly, yeast cultures were grown in 500 mL of YPD to an OD600 of 0.7-0.9 before crosslinking with 1% formaldehyde (Fisher Scientific, BP531-500) at room temperature for 15 min and quenched with 125 mM glycine at room temperature for 5 min. Crosslinked cells were collected by centrifugation and washed twice with ice-cold ddH2O. Yeast cell wall digestion was then performed by resuspending cell pellets in 39 mL Buffer Z (1 M sorbitol and 50 mM Tris-HCl pH 7.4) containing 10 mM β-mercaptoethanol and 10 mg Zymolyase 100T (US Biological, Z1004) and incubating at 30˚C with agitation (20-40 min). When digestion was completed, the resulting spheroplasts were pelleted by centrifugation at 3,000 g for 5 min at 4°C and gently resuspended in 2.5 mL of NP buffer (1 M sorbitol, 50 mM NaCl, 10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 µM spermidine (Sigma-Aldrich, 85558), 1 mM β-mercaptoethanol and 0.075% NP-40) before being exposed to MNase (Worthington, LS004798) digestion titration to generate the appropriate size of chromatin fragments as follow. 700 µl of spheroplasts were incubated with 4.5, 9, 18, and 36 units of MNase (resuspended in 10 mM Tris-HCl pH 7.4 and 30% glycerol) at 37˚C for 20 min and reaction was stopped by adding 10 mM EDTA. To ensure IP conditions, 200 µL of adjusting buffer (50 mM HEPES-KOH pH7.5, 140 mM NaCl, 1% Triton X-100, and 0.1% Na-deoxycholate) were added to the digested chromatin sample. To determine the appropriate condition of MNase digestion, 45 µL (5%) of each MNase digested sample were taken and incubated with 5 μL of 10X TE/SDS at 65˚C for 16 hr to reverse crosslinking. Samples were then treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 100 % ethanol. Precipitated DNA was resuspended in 30 μL of TE, treated with 1µL of 10 mg/mL RNAse A at 37°C for 1 hr before being analyzed by electrophoresis on a 1.8% TAE-agarose gel stained with ethidium bromide. The digested samples were also qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA Kit (Agilent Technologies, 5067-4626). The condition giving approximately 65% mono-, 25% di- and 10% tri-nucleosomes was subjected to IP as follow. 45µL (5%) were saved as Input sample and the remaining digested chromatin was incubated overnight with the appropriate antibody-coupled Dynabeads as per our standard ChIP protocol (see above). Beads were then washed and samples reverse crosslinked, treated with RNase A, and subsequently subjected to Proteinase K before being twice phenol/chloroform/isoamyl alcohol extracted followed by precipitation with NaCl and ethanol. Precipitated DNA was resuspended in 50 μL of TE before being used in sequencing libraries.  Before proceeding with MNase-ChIP-seq libraries, ChIP and Input samples were subjected to a 2X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions. Samples were eluted with 50 μL of Elution buffer (10 mM Tris pH 8.0) and quantified/qualified on an Agilent 2100 Bioanalyzer instrument using the High Sensitivity DNA kit.  1 ng of Input DNA and 40 μL of ChIP DNA were used for library preparation as follow. The ends of DNA were repaired by incubating in 70 μL of 1X NEBuffer 2 containing 0.6 units of T4 DNA polymerase (NEB, M0203L), 2 units of T4 polynucleotide kinase (NEB, M0201S), 0.09 nM dNTPs and 0.045 μg/μL of BSA at 12˚C for 30 min. Repaired DNA was then subjected to a 2X cleanup using KAPA Pure Beads before dA-tailing as follow. Beads containing the repaired DNA were resuspended in 50 μL of 1X NEBuffer 2 containing 0.1 mM dATP and 25 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M), and incubated at 37˚C for 30 min. After a 2X cleanup with KAPA Pure Beads, dA-tailed DNA was ligated to index adapters (Roche, SeqCap Adapter kit A (07141530001) and SeqCap Adapter kit B (07141548001)) as follow. The beads were resuspended in 45 μL of 1X Ligase buffer containing 8 nM of adapter and 2.5 units of T4 DNA ligase and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1X cleanup with KAPA Pure Beads. Libraries were PCR-amplified with 10-12 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and cleaned up using 1X KAPA Pure Beads. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library was pooled (12 libraries per pool) and subjected to sequencing on Illumina HiSeq 4000 or NovaSeq 6000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID:Adapter 14 from SeqCap Adapter kits A and B (Roche, 07141530001 and 07141548001)
MNase-ChIP-Seq