title: DHO2_4D

city: Sacramento

first: Dennis

middle: J.

last: Hartigan-O'Connor

state: CALIFORNIA

address: 3146 One Shields Avenue, Tupper Hall

department: Medical Microbiology and Immunology

institute: University of California, Davis

data_processing:
STAR version 2.7 was used to align sequences to Macaca_mulatta.Mmul_10.dna.primary_assembly and annotated with Macaca_mulatta.Mmul_10.99.gtf file
Genome_build: Mmul_10
Supplementary_files_format_and_content: raw_counts.csv is a matrix containing features annotated with Ensembl ID and raw gene counts for each sample.

source_name: Antigen presenting cell

extract_protocol:
Norgen Total RNA Purification Micro Kit
Lexogen QuantSeq 3' mRNA-Seq Library Prep Kit

treatment_protocol: flow cytometry sorting into RNAlater

tag: rhcmv status

value: neg

value: F

tag: animal id

tag: age (year)

tag: fragmentation time (sec)


title: DHO2_5D

status: Public on Jul 02 2021

type: SRA

country: USA

library_source: transcriptomic

molecule: total RNA

organism: Macaca mulatta

tag: Sex


title: RNA-seq_3W3

city: Grand Forks

first: Junguk

last: Hur

state: ND

address: 1301 N. Columbia Rd. Stop 9037

department: Biomedical Sciences

institute: University of North Dakota

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adapter sequence and low-quality sequence using Trimmomatic
Reads were mapped to the mm10 reference genome using Hisat2 sequence analysis program to identify transcripts.
Count values were calculated using featureCounts
Counts were normalized by DESeq2 and rlogTransformed
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include rlogTransformed values for each sample in the matrix

organism: Mus musculus

source_name: Microglia Enriched Brain homogenate

growth_protocol: Mice were provided with food and water ad libitum and housed in a 12-hour light/dark cycle.

extract_protocol:
The AllPrep DNA/RNA MiniKit (Qiagen; Germantown, MD; Product ID: 80204) was used to individually extract RNA and DNA from each flash-frozen lysate sample. All RNA and DNA samples were stored at -80°C for later use.
RNA libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA).

treatment_protocol: Microglia extraction was performed on brains dissected from the mice involved in this study. This protocol was adapted from Lee and Tansey (PMID:23813365).

tag: genotype

value: WT

tag: age

value: 3 months

tag: tissue

value: Microglia Enriched Brain homogenate

tag: background strain

tag: group


title: DHO2_3D


title: Input1_JRY10156

city: Buffalo

first: Laura

middle: N

last: Rusche

state: NY

address: 109 Cooke Hall, UB North/Amherst Campus

department: Biological Sciences

institute: State University of New York at Buffalo

library_source: genomic

data_processing:
Paired-end sequence reads were processed to remove 3’ adapter sequences using Cutadapt 2.10 with the parameters --nextseq-trim=20 and -m 10.
T. delbrueckii genome index was built using Bowtie2/2.3.4.1.
ChIP- seq reads for both single-end and paired-end sequencing were aligned to the T. delbrueckii genome index using Bowtie2/2.3.4.1.
ChIP peaks were called using MACS2/2.2.7.1 with the parameters: --nomodel was used for both, --extsize 200 was used for single-end reads only, -f BEDPE was used for paired end reads
Genome_build: CBS1146 (GCA_000243375.1)
Supplementary_files_format_and_content: bigWig

molecule: genomic DNA

organism: Torulaspora delbrueckii

source_name: Yeast

growth_protocol: Yeast cells were grown at 30˚C in YPD medium containing 1% yeast extract, 2% peptone, and 2% glucose.

extract_protocol:
Cells were grown in YPD overnight. Following day, cells were diluted at OD600 0.3, grown at 30˚C, and harvested at an OD600 of ∼1.
Libraries were prepared using Takara Bio SMARTer ThruPlex DNA seq kit

treatment_protocol: Cells were cross-linked for 1 hour in 1% formaldehyde. After cell lysis, chromatin was sonicated. Immunoprecipitation was conducted with 10μL Protein A agarose beads and 4 µl of anti MYC (Millipore 06-549) or anti V5 antibody (Millipore AB3792) antibody.

tag: strain

tag: epitope tag

tag: chip antibody


title: T2

city: Göttingen

first: Shiv K.

last: Singh

address: Robert-Koch-Str. 40

department: Gastroenterology and Gastrointestinal Oncology

country: Germany

institute: Univeristy Medical Center

description: 10 ng/ul TNFα (18h)

data_processing:
Raw reads were quality-checked using FastQC v0.11.5
Alignment against hg38 human reference genome was done by using STAR v2.5.2b
Aligned reads were assigned to genes using HTSeq-count version 0.10.0
DESeq2 was utilized for differential gene expression analysis
Genome_build: hg38
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

organism: Homo sapiens

source_name: Pancreatic cancer

growth_protocol: CAPAN1 cells were cultured in RPMI 1640 supplemented with 10% FCS and 1% P/S.

extract_protocol:
RNA was harvested using Trizol reagent
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Cells were seeded in 6-well plate 24 h prior to treatment. Next day cells were exposed to 10 ng/ul TNFα for 18 h, aqua dest was used as a control.

tag: cell line

tag: treatment

value: 10 ng/ul TNFalpha

tag: time point


title: T1

description: 10 ng/ul TNFα (18h)


title: Subject 23 G2b-04M

type: RNA

city: Kansas City

first: Zohreh

last: Talebizadeh

state: MO

address: 2401 Gillham Road

institute: Children's Mercy Hospital

hyb_protocol: Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies). On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to target microRNA (from miRBase, http://miRBase.org) or other RNA (control or customer defined sequences) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using PGR (photogenerated reagent) chemistry. The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 uL 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 °C.

scan_protocol: The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 mL 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 °C.  After RNA hybridization, tag-conjugating Alexa Fluor®546 dye was circulated through the microfluidic chip for dye staining.  Fluorescence images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics).

data_processing: Data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS filter (Locally-weighted Regression) (2). (2) Bolstad, B. M., Irizarry, R. A., Astrandand, M., Speed, T. P. (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinfo. 19, 185-193.

source_name: blood microRNA

label_protocol: Microarray assay was performed using a service provider (LC Sciences).  Total RNA sample (2 µg) were 3’-extended with a poly(A) tail using poly(A) polymerase.  An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining.  Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies) (1). On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to target microRNA (from miRBase, http://mirbase.org) or other RNA (control or customer defined sequences) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate.  The detection probes were made by in situ synthesis using PGR (photogenerated reagent) chemistry.  (1) (a) Gao, X., Gulari, E., and Zhou, X. (2004) In situ synthesis of oligonucleotide microarrays. Biopolymers 73, 579-596; (b) Zhu, Q., Hong, A., Sheng, N., Zhang, X., Jun, K.-Y., Srivannavit, O., Gulari, E., Gao, X., and Zhou, X. (2007) Microfluidic biochip for nucleic acid and protein analysis. in Methods Mol. Biol. Ed. Rampal, J. B. 382:287-312.

extract_protocol: Qiagen miRNeasy kit was used for total RNA extraction following manufacturer's protocol.

value: maternal blood

value: Female

tag: sert genotype

value: SS

tag: prenatal stress level

value: Low


title: RNA-seq_3W1


title: RRBS_13H13

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then processed using the bowtie2 sequence analysis programs. Reads were mapped to the ensembl reference human genome.
To analyze the sequence data, a reference genome is created that contains only the 36 base pairs adjacent to every MspI site and every C in those sequences is changed to T. A converted sequence read file is then created by changing each C in the original sequence reads to a T. The converted sequence reads are aligned to the converted reference genome, and only reads that map uniquely to the reference genome are kept. Once reads are aligned the percent methylation is calculated for each CpG using the original sequence reads. The percent methylation and number of reads is reported for each CpG.
Methylation percentage for each CpG sites were calcualted by methylKit
Genome_build: mm10
Supplementary_files_format_and_content: Tab delimited text files including methylation percentage for each sample in the matrix for 3 and 13-month old animal.

extract_protocol:
The AllPrep DNA/RNA MiniKit (Qiagen; Germantown, MD; Product ID: 80204) was used to individually extract RNA and DNA from each flash-frozen lysate sample. All RNA and DNA samples were stored at -80°C for later use.
RRBS samples were prepared at the University of Michigan Epigenetics Core facility (PMID: 25742437)

value: microglia enriched brain homogenate

value: HEM

value: 13 months


title: DHO2_2D

tag: rhcmv status

value: M

tag: animal id


title: Subject 20 G2a-27M

value: LL

value: High


title: Subject 22 G2a-43M


title: Subject 25 G2b-02M


title: DHO2_6D

tag: age (year)

tag: fragmentation time (sec)


title: DHO2_1D

tag: Sex


title: C2

value: aqua dest


title: RNA-seq_3H7

tag: genotype

tag: age


title: Subject 24 G2b-41M

tag: tissue


title: RNA-seq_3H4

tag: background strain

tag: group


title: C1


title: RNA-seq_3H3


title: NP4

city: Solna

first: Christopher

last: Tibbitt

state: Stockholms län

address: C7 Biomedicum

department: MTC

country: Sweden

institute: Karolinksa Institutet

data_processing:
To generate the cell (barcode)-gene matrices, raw sequencing data were demultiplexed to individual fastq files, then aligned and quantified by Cell Ranger v3.1 (10xGenomics) under default setting. Human genome GRCh38 (UCUS) was adopted as reference. Ambient RNA in raw cell-gene matrices were cleaned based on the nUMI-barcode saturation curves.  Cell clustering and differentially expressed gene profile:  The R package Seurat v3.1 was used under R v3.6 for scRNA-seq data trimming, analyzing, and visualization (Butler et al., 2018; Stuart et al., 2019).
Genome_build: Genome_build: hg19 –GRCh38

source_name: Nasal Polyp

extract_protocol:
Nasal polyp tissues were cleared of connective tissue, cut into fine pieces and then passed through 100uM sieves. Cell pellets were resuspended in IMDM supplemented with penicillin/streptomycin, glutamine, 2- mercaptoethanol (all from Invitrogen), and 10% heat-inactivated FCS (Sigma).
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: phenotype

value: Chronic rhinosinusitis


title: Subject 21 G2a-30M


title: DHO2_5C

source_name: Natural killer cell

value: neg

value: F


title: RNA-seq_3H1

value: 3 months


title: DHO2_3C


title: NP3


title: NP2


title: RNA-seq_13W9

value: WT

value: Microglia Enriched Brain homogenate


title: DHO2_4C


title: Subject 19 G2a-58M

value: maternal blood


title: RNA-seq_13W8


title: DHO2_2C


title: C3


title: Subject 16 G1b-48M

value: Female

value: LS


title: Subject 18 G2a-13M


title: NP1


title: DHO2_1C


title: RNA-seq_13W7


title: Subject 15 G1b-46M


title: 5168-Tr1-2

city: Stanford

first: Molly

last: Uyeda

state: CA

address: 265 Campus Drive

institute: Stanford University

data_processing:
Skewer (Jiang, H., Lei, R., Ding, S. W., & Zhu, S. (2014). Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC bioinformatics, 15(1), 1-12).
STAR dual pass (Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., ... & Gingeras, T. R. (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics, 29(1), 15-21).
Genome_build: GRCh38
Supplementary_files_format_and_content: all_stats_and_counts.txt: Tab-delimited text file includes raw counts for each Sample.

source_name: circulating Tr1

growth_protocol: Peripheral blood mononuclear cells were isolated from a buffy coat and subject to FACS to subisolate Tr1 and non-Tr1 memory cells.

extract_protocol:
20,000 cells were lysed in Trizol-LS. Phenol chloroform extraction.
Nugen ovation RNAseq system V2 followed by NEBNEXT Ultra DNA for Illumina kit.

tag: cell type

value: circulating Tr1

tag: donor

tag: disease status

value: healthy


title: RNA-seq_13W6


title: Subject 17 G1b-53M


title: DHO2_6C


title: DHO2_6B

source_name: CD8+NKG2A/C- T cell


title: Subject 13 G1b-32M


title: Subject 14 G1b-45M


title: 5168-Tr1-1


title: Subject 12 G1b-31M


title: RNA-seq_13H13

value: HEM


title: DHO2_5B


title: Subject 11 G1b-28M


title: 5168-DN-2

source_name: circulating non-Tr1 T memory cells

value: circulating non-Tr1 T memory cells


title: DHO2_4B


title: RNA-seq_13H12

value: 13 months


title: 5168-DN-1


title: 2857-Tr1-2


title: RNA-seq_13H11


title: 2857-Tr1-1


title: RNA-seq_13H7


title: Subject 10 G1b-26M


title: RNA-seq_13H8


title: DHO2_2B

value: M


title: DHO2_1B


title: Subject 9 G1b-23M

value: High


title: DHO1_6D

value: pos


title: RNA-seq_13H4


title: 2857-DN-2


title: 2857-DN-1


title: Subject 8 G1b-22M


title: DHO1_5D


title: RNA-seq_13H3


title: RNA-seq_13H1


title: Subject 7 G1b-12M


title: DHO1_4D


title: 2858-DN-2


title: 2858-Tr1-2


title: Subject 6 G1a-05M

value: SS


title: DHO2_3B

value: neg


title: OVX6024_inf

city: Kolkata

first: Pallob

last: Kundu

state: West Bengal

address: P 1/12 CIT Scheme VIIM

department: Division of Plant Biology

country: India

institute: Bose Institute

data_processing:
All the 150 bp paired end raw reads (Illumina HiSeq) were quality checked for low quality bases and adapter  sequences (Processing of raw reads). Quality check was done using perl scripts.
All the processed reads were aligned to Solanum lycopersicum genome using:  Tophat2
Transcripts were assembled using Cufflinks and the relative abundances of these transcripts were estimated based  on how many reads support each one, taking into account biases in library preparation protocols. Cuffdiff was used for performing differential gene expression study.
Genome_build: SL3.0

organism: Solanum lycopersicum

source_name: Leaf

growth_protocol: Grown in synthetic soil (Soilrite mix Tc. Keltech Energies Limited) in plastic pots and maintained at 16hr light/8hr dark cycles at 25° C temperature and 70% humidity.

extract_protocol:
Leaf tissue was frozen in liquid Nitrogen and total RNA was extracted using TRIzol reagent. 1ug of Total RNA was used to enrich mRNA using NEB Magnetic mRNA Isolation Kit.
The transcriptome library was prepared using NEB ultraII RNA library prep kit and sequenced using Illumina Next Seq 500 paired end technology using standard protocol

treatment_protocol: 1.5 month old wild type and OVX6024 plants were drop inoculated on the adaxial surface with a suspension of spores and mycelia (10^6 CFU/ml water) of actively growing A. solani. The incubation period was 48 hrs, before tissue was harvested for RNA extraction.

tag: variety

value: Pusa Ruby

value: Leaf

tag: treatment

value: A. solani infected


title: WT_inf


title: DHO1_2D


title: DHO1_3D

value: F


title: DHO1_1D


title: Subject 5 G1a-20M

value: maternal blood

tag: sert genotype

tag: prenatal stress level


title: OVX6024.2

value: untreated


title: Primed_BRG1_HET

city: Saint Louis

first: Bo

last: Zhang

state: Missouri

address: 4515 McKinley Research Bldg 03212

department: Developmental Biology

institute: Washington University School of Medicine

data_processing:
Raw reads were processed by AIAP v1.1 (https://www.biorxiv.org/content/10.1101/686808v1). Briefly, reads were quality trimmed by cutadapt. Trimmed reads were aligned by bwa. Aligned reads were quanitfied using methylQA density and peaks called with macs2. Signal density files normalized to 10 million reads were also generated.
Genome_build: hg38
Supplementary_files_format_and_content: BigWig density file and peaks call bed file

source_name: hPSC

growth_protocol: H9 primed hESCs were cultured in mTeSR Plus on Matrigel hESC-Qualified Matrix coated wells and H9 naïve hESCs were maintained on mitomycin C inactivated MEFs feeder cells in PXGL media consisting of N2B27 supplemented with 1 μM PD0325901, 2 μM XAV939, 2 μM Gö6983, and 20 ng/mL human LIF. All naïve hESC experiments were conducted in 5% O2, 5% CO2. For ATAC-seq analysis of re-priming, naïve hESCs were seeded on a MEF feeder layer and harvested four days after transfer to mTeSR Plus media. Primed and re-primed cultures were conducted in 20% O2 and 5% CO2.

extract_protocol:
Cells were harvested by TrypLE Express dissociation and centrifuged at 500 RCF for 5 min at 4 ℃. After aspirating supernatant, cells were washed once with cold PBS. Cell pellets were resuspended in 100 ul ATAC-seq RSB [10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2 in water] consisting of 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin by pipetting up and down and incubating on ice for 3 min. After lysis, 1 mL of ATAC-seq RSB containing 0.1% Tween-20 was added and inverted with the lysis reaction. Then, nuclei were pelleted by centrifugation at 800 RCF for 5 min at 4 ℃. Supernatant was removed, and the nuclei were resuspended in 20 µL 2x TD buffer [20 mM Tris pH 7.6, 10 mM MgCl2, 20% Dimethyl Formamide]. 50,000 counted nuclei were then transferred to a tube with 2x TD buffer filled up to 25 µL. 25 µL of transposition mix [2.5 µL Transposase (100 nM final) (Illunina, 20034197, 16.5 µL PBS, 0.5 µL 1% digitonin, 0.5 µL 10% Tween-20, and 5 µL water) was added. Transposition reactions were mixed and incubated for 30 min at 37℃ with gentle tapping every 10 min. Reactions were cleaned up with the Zymo DNA Clean and Concentrator-5 kit (Zymo Research, D4014).
The ATAC-seq library was amplified for nine cycles on a PCR machine. The PCR reaction was purified with Ampure XP beads (Beckman Coulter, A63880) using double size selection following the manufacturer’s protocol, in which 27.5 µL beads (0.55X sample volume) and 50 µL beads (1.55X sample volume) were used based on 50 µL PCR reaction. The ATAC-seq libraries were quantitated by Qubit assays and sequenced by an Illumina NextSeq platform. QC and analysis on ATAC-seq libraries was performed using AIAP (Liu et al., 2019).

value: hPSC

tag: genotype/variation

value: HET

tag: iPSc source

value: Human Embryonic Stem cells

tag: passage


title: OVX6024.1


title: DHO1_6C

tag: rhcmv status

tag: Sex

tag: animal id

tag: age (year)

tag: fragmentation time (sec)


title: Primed_BRG1_WT

value: WT


title: rRPE3

city: Baltimore

first: Seth

last: Blackshaw

state: MD

address: 733 N Broadway

department: Neuroscience

institute: Johns Hopkins University

data_processing:
Read trimming with Trim Galore v0.6.1 and Cutadapt v1.18
Alignment to GRCg6a with STAR v2.7.3
Transcript quantification with Stringtie v2.0.4 and Ensembl 98
Gene count normalization with DESeq2 v1.22.2
Genome_build: GRCg6a
Supplementary_files_format_and_content: DESeq2 normalized gene counts

molecule: polyA RNA

organism: Gallus gallus

source_name: E4 RPE 6 hours post retinectomy and FGF2 treatment (reprogrammed RPE; rRPE)

extract_protocol:
Embryos were collected and infiltrated at 4°C with a sucrose gradient followed by 2:1 25% sucrose:OCT compound (Sakura Finetek, Torrance, CA) for 1 h and snap frozen in dry ice and methylbutane. Cryosections (12μm) were collected on PEN Membrane Frame Slides (ThermoFisher Scientific) followed by fixation in 70% ethanol at -20°C for 30 sec. Slides were rehydrated in 4°C DEPC-treated water for 30s, stained with hematoxylin (Sigma, St. Louis, MO) for 10 sec, and dehydrated in a 70%, 95%, and 100% ethanol gradient for 30 seconds each. LCM was performed using a Veritas Laser Capture Microdissection system and software to collect RNA samples for library preparation. RPE was isolated from sections using IR capture and UV dissection on CapSure HS LCM Caps (ThermoFisher Scientific, Waltham, MA), and total RNA extraction was performed using PicoPure RNA Isolation Kit (Arcturus, Applied Biosystems, Foster city, CA), including a treatment with DNase I.
Total RNA was processed for library construction by Cofactor Genomics (http://cofactorgenomics.com, St. Louis, MO) according to the following procedure. Briefly, total RNA was reverse-transcribed using an Oligo(dT) primer, and limited cDNA amplification was performed using the SMARTer® Ultra® Low Input RNA Kit for Sequencing – v4 (Takara Bio USA, Inc., Mountain View, CA ). The resulting full-length cDNA was fragmented and tagged, followed by limited PCR enrichment to generate the final cDNA sequencing library (Nextera® XT DNA Library Prep, Illumina, San Diego, CA). Libraries were sequenced as single-end 75 base pair reads on an Illumina NextSeq500 following the manufacturer's instructions.

tag: developmental stage

value: Embyonic day 4

tag: tissue

value: retinal pigment epithelium (RPE)


title: rRPE2


title: WT


title: DHO1_5C

value: M


title: Subject 3 G1a-44M

value: Female

value: High


title: Subject 4 G1a-51M

value: SS


title: Subject 1 G1a-10M


title: rRPE1


title: met1MORC7-FLAG_RNAseq_rep-3

city: Los Angeles

first: Zhenhui

last: Zhong

address: 610 Charles E Young Dr East

department: Department of Molecular, Cell and Developmental Biology

institute: University of California, Los Angeles

data_processing:
All libraries were sequenced at a length of 50 bps with HiSeq 2500 or NovaSeq 6000 platforms following manufacturer’s instructions (Illumina). Raw reads were aligned to the Arabidopsis reference genome (TAIR10) with Bowtie2 (v2.1.0)43, allowing only uniquely mapped reads with perfect matches. Duplicated reads were removed with Samtools (v1.9)44. Peaks were called using MACS2 (v2.1.1)45.
BS-seq reads were mapped to TAIR10 reference genome by bsmap (v2.90) with allowing 2 mismatches and 1 best hit (-v 2 -w 1)46. Reads with three or more consecutively methylated CHH sites were considered as non-converted reads and removed from the analyses. DNA methylation levels were calculated by #C/ (#C + #T). Differential Methylated Regions (DMRs) were called by methdiff function with every 100bp bin for where the difference in CG, CHG, and CHH methylation are at least 0.4, 0.2, and 0.1, respectively.
Genome_build: tair10
Supplementary_files_format_and_content: bigWig

organism: Arabidopsis thaliana

growth_protocol: Arabidopsis plants of the Columbia-0 (Col-0) ecotype were used in this study. All plants were grown under green house condition (16 h light/8 h dark, 22C). Floral tissues were collected from 5-6 week plants

extract_protocol:
ChIP seq: Samples were crosslinked in vitro. Nuclei were prepared with Nuclei Isolation Buffer (50 mM HEPES, 1 M sucrose, 5 mM KCl, 5 mM MgCl2, 0.6% Triton X-100, 0.4 mM PMSF, 5 mM benzamidine, cOmplete EDTA-free Protease Inhibitor Cocktail (Roche)). Chromatin was sheared with Bioruptor Plus (Diagenode) for 20 minutes with 30 s on and 30 s off and incubated with anti-FLAG M2 (Sigma) antibody overnight at 4◦C. Chromatin bound proteins were immunoprecipitated with Protein A and Protein G magnetic Dynabeads (Invitrogen) for 2 hours at 4◦C. DNA fragments were precipitated with EtOH overnight at -20◦C. Libraries were prepared with Ovation Ultra Low System V2 kits following the manufacturer’s instructions. BS seq: DNA from floral tissues were extracted with cetyl trimethylammonium bromide (CTAB)-based method. RNA was removed with PureLink RNase A (Invitrogen). 300 ng of DNA was sheared to 200bp with a Covaris S2 (Covaris). RNA seq: Floral tissues from 5-6 week old seedlings were used for RNA extraction with Zymo Direct-zol RNA MiniPrep kit (Zymo Research) following manufacturer’s instructions.
ChIP seq: Libraries were prepared with Ovation Ultra Low System V2 kits following the manufacturer’s instructions. BS seeq: 300 ng of DNA was sheared to 200bp with a Covaris S2 (Covaris). Libraries were prepared with the Epitect Bisulfite Conversion kit (QIAGEN) and the Ovation Ultralow Methyl-seq kit (NuGEN) following the manufacturer’s instructions. RNA seq: 1 ug of total RNA was used for library preparation with TruSeq Stranded mRNA kit (Illumina). Libraries were sequenced on HiSeq 2500 or NovaSeq 6000 (Illumina).

value: floral tissue

value: 5-6 weeks

tag: strain


title: DHO1_4C

value: pos


title: 2858-DN-1

tag: cell type

value: circulating non-Tr1 T memory cells

tag: donor

value: healthy


title: Re-Priming_BRG1_KO_rep3

value: KO


title: DHO1_3C

value: F


title: Re-Priming_BRG1_KO_rep2

value: hPSC


title: trRPE3

source_name: E4 RPE 6 hours post retinectomy (transiently reprogrammed RPE; t-rRPE)


title: met1MORC7-FLAG_RNAseq_rep-2


title: trRPE2


title: Re-Priming_BRG1_KO_rep1


title: Re-Priming_BRG1_HET

value: HET


title: trRPE1


title: Subject 2 G1a-25M

value: maternal blood


title: Re-Priming_BRG1_WT

value: WT

value: Human Embryonic Stem cells


title: DHO1_1C


title: MORC7-FLAG_RNAseq_rep-3


title: DHO1_2C


title: MORC7-FLAG_RNAseq_rep-2


title: Naive_BRG1_KO_rep2


title: RPE3

source_name: intact embryonic day 4 (E4) RPE

value: Embyonic day 4

value: retinal pigment epithelium (RPE)


title: Naive_BRG1_KO_rep1


title: DHO1_6B


title: RPE2


title: DHO1_5B

value: M


title: MORC7-FLAG_RNAseq_rep-1


title: RPE1


title: DHO1_4B


title: RNAse treated OriSSDS Mouse ESCs

city: Bethesda

first: Kevin

last: Brick

address: 5/205 Memorial Drive

department: GBB

institute: NIDDK

description: Cultured Embryonic Stem Cells

data_processing:
Library strategy: Ori-SSDS
----------ALIGNMENT----------
Sequencing reads from Ori-SSDS experiments were aligned to the mouse mm10 genome using bwa 0.7.12 and the single-stranded DNA pipeline (Brick et al. 2018; Khil et al. 2012). Only fragments derived unambiguously from ssDNA (ssDNA_type1) were used in subsequent analyses. ssDNA fragments where either read1 or read2 had mapping quality <30 were discarded. Fragments from the mitochondrial chromosome and fragments in blacklisted regions of the genome were also discarded. Duplicate fragments were subsequently discarded.
----------PEAK CALLING----------
We used the MACS algorithm for peak calling. To assure that data derived from Ori-SSDS fit the MACS peak model, we performed two operations:
1. The strand orientation of fragments was reversed prior to peak calling.
2. The data was passed to MACS as ssDNA fragment BED files (from SSDS pipeline).
Peak calling was performed for each Ori-SSDS sample using MACS 2.1.2 and the following parameters : -g mm -q 0.001 -extsize 2000 --nomodel --nolambda.
For origin calls from individual samples, peaks that did not exhibit the expected Crick-Watson asymmetry were discarded (see paper Figure 1A).
The high-confidence origin set was defined by merging origin calls from wt_Rep1,2,3 and ESC_Rep1. Origin strength was calculated in each sample using a method analogous to that for DMC1-SSDS hotspot mapping(Brick et al. 2018). Briefly, each origin is recentred to the midpoint of the Watson and Crick fragment distributions. Signal is estimated as the Crick-strand signal to the left of the origin center + the Watson-strand signal to the right of the origin center. The background is estimated by counting the remaining fragments at the left and right edges of origins (15%); Watson-strand fragments to the left of the origin center, Crick-strand fragments to the right. This background is then extrapolated to the entire origin and subtracted from the signal. Following this process, origins found in just one sample and narrow origins (<7 Kb) origins that did not display the expected Crick/Watson asymmetry (see paper Figure 1A) in any sample were discarded.
----------BIGWIG files----------
Duplicate fragments were removed from the ssDNA type1 BED file. separate BED files were created from +-strand, --strand and all fragments. Each BED file was converted to a BAM using "bedtools bedtobam" and sorted to match the genome reference fasta file. The bamCoverage tool from deeptools v3.0.1 was used to cenerate a BIGWIG file with the following arguments ( --outFileFormat bigwig --binSize 150 -p max --ignoreForNormalization chrX chrM chrY --normalizeUsing RPKM).
Genome_build: mm10
Supplementary_files_format_and_content: BIGWIG, BEDGRAPH

source_name: RNAse control for Leading strand capture and single stranded DNA sequencing for mouse cultured ESCs (Sample 2)

growth_protocol: Cells were cultured as recommended by Jackson labs in the presence of mitotically inactive MEF feeder cells. Exponentially growing cells were collected and 4x107 were used for origin detection.

extract_protocol:
Cells were suspended in 8 ml of DNAzol (Thermo-Fisher catalog number 10503027). The DNA was precipitated with 5 ml of EtOH and then spooled with a pipette tip and rinsed gently twice with EtOH 75% by sequential transfer into 15 ml tubes. DNA was air-dried for 5 min and then resuspended in 500 μl of 1X TEN buffer (Tris 10mM pH=8; EDTA 1mM; NaCl 100mM) + 80 U of RNAseOUT (40 U/μl) at 4C for at least 24h.
DNA was denatured at 95°C for 5 min, chilled on ice for 5 min and size-fractionated on 5–30% (w/v) linear sucrose gradients in TEN buffer in a Beckman Ultracentrifuge SW45 rotor for 18 h at 26,000 g at 20°C. 500 ul fractions were collected from the top of the gradient, precipitated by adding 1 ml of EtOH 100 % and 50 μl of NaOAc 3M pH 5.2 and resuspended in 25 μl Tris Buffer pH=8. Fractions were analyzed in a denaturing (50 mM NaOH, 1 mM EDTA) 1.2% agarose gel and the ones containing ssDNA ranging in size from 800 to 2000 nt were pooled. SNS were heat-denatured for 5 min at 95°C and chilled on ice for 5 min. DNA was incubated with a mix of RNAseA/T and RNAseI in 1X TEN buffer (Tris 10mM pH=8; EDTA 1mM; NaCl 100mM) for 30 min at 37°C. DNA is phosphorylated by Polynucleotide Kinase in 150 ul reactions in PNK Buffer (100 U of PNK ; 1X PNK buffer ; 1 mM ATP; 40U RNAseOUT). Reactions were incubated for 30 min at 37°C and heat-inactivated at 75°C for 15 min. DNA was extracted with Phenol/Chloroform, EtOH precipitated and resuspended in 50 μl Tris Buffer pH=8. Samples were heat-denatured again for 5 min and digested with lambda-exonuclease in 100 μl of custom lambda buffer (67 mM glycine-KOH pH= 8.8, 2.5 mM MgCl2, 50 μg/ml BSA) with 2.5 U lambda-exonuclease/μg DNA (Thermo Scientific) and 80U RNAseOUT and incubated overnight at 37°C. Reactions were inactivated for 10 min at 75°C and DNA was purified with MinElute columns (QIAGEN), as indicated by the instruction manual. RNA primers were hydrolyzed by incubating the eluate for 30 min at 37°C in 0.25 N NaOH, followed by neutralization with acetic acid and purified again with MinElute columns.

value: Cultured Embryonic Stem Cells

tag: experiment type

value: Origin of replication mapping using leading strand capture and Single Stranded DNA Sequencing (Ori-SSDS)

tag: dna content

tag: s-phase sub-stage

tag: cell-type

tag: positive selection marker

tag: negative selection marker

tag: antibody


title: met1MORC7-FLAG_ChIP_rep-2

value: floral tissue

value: anti-FLAG M2 (Sigma)

tag: target protein


title: DHO1_3B

tag: rhcmv status

value: pos

tag: Sex


title: met1MORC7-FLAG_ChIP_rep-1

value: 5-6 weeks


title: Naive_BRG1_WT

tag: genotype/variation

tag: iPSc source

tag: passage


title: Naive_BRG1_HET


title: CD11c+ DCs treated with PBS_rep3

city: Suita

first: Daisuke

last: Okuzaki

state: Osaka

address: Yamadaoka 3-1

department: Immunology Frontier Research Center

country: Japan

institute: Osaka univ.

description: CD11c+ DCs treated with PBS_rep3

data_processing:
Sequencing was performed on an Illumina HiSeq 2500 platform in a 75-base single-end mode.
Illumina Casava1.8.2 software used for basecalling.
Sequenced reads were mapped to the mouse reference genome sequences (mm10) using TopHat v2.0.13 in combination with Bowtie2 ver. 2.2.3 and SAMtools ver. 0.1.19.
The fragments per kilobase of exon per million mapped fragments (FPKMs) was calculated using Cuffnorm version 2.2.1.
Genome_build: mm10

source_name: CD11c+ dendritic cells

extract_protocol:
Total RNA was extracted using the RNeasy Mini Kit (Qiagen). Then RNA was treated with DNase1 (Invitrogen). We cleaned up RNA with RNeasy Mini Kit again.
Library preparation was performed using a TruSeq stranded mRNA sample prep kit (Illumina, San Diego, CA) according to the manufacturer’s instructions.

treatment_protocol: Splenic cells were extracted and CD11c+ dendritic cells (DCs) were sorted by autoMACS.

tag: treatment

value: treated with PBS

value: CD11c+ dendritic cells


title: CD11c+ DCs treated with PBS_rep2

description: CD11c+ DCs treated with PBS_rep2


title: DHO1_2B

tag: animal id

tag: age (year)

tag: fragmentation time (sec)


title: MORC7-FLAG_anti-HA_ChIP

tag: tissue

tag: developmental stage

tag: strain


title: met1MORC7-FLAG_RNAseq_rep-1


title: OriSSDS Mouse ESCs Sample 2

description: Cultured Embryonic Stem Cells

source_name: Leading strand capture and single stranded DNA sequencing for mouse cultured ESCs (Sample 2)

extract_protocol:
Cells were suspended in 8 ml of DNAzol (Thermo-Fisher catalog number 10503027). The DNA was precipitated with 5 ml of EtOH and then spooled with a pipette tip and rinsed gently twice with EtOH 75% by sequential transfer into 15 ml tubes. DNA was air-dried for 5 min and then resuspended in 500 μl of 1X TEN buffer (Tris 10mM pH=8; EDTA 1mM; NaCl 100mM) + 80 U of RNAseOUT (40 U/μl) at 4C for at least 24h.
DNA was denatured at 95°C for 5 min, chilled on ice for 5 min and size-fractionated on 5–30% (w/v) linear sucrose gradients in TEN buffer in a Beckman Ultracentrifuge SW45 rotor for 18 h at 26,000 g at 20°C. 500 ul fractions were collected from the top of the gradient, precipitated by adding 1 ml of EtOH 100 % and 50 μl of NaOAc 3M pH 5.2 and resuspended in 25 μl Tris Buffer pH=8. Fractions were analyzed in a denaturing (50 mM NaOH, 1 mM EDTA) 1.2% agarose gel and the ones containing ssDNA ranging in size from 800 to 2000 nt were pooled. SNS were heat-denatured for 5 min at 95°C and chilled on ice for 5 min and DNA is phosphorylated by Polynucleotide Kinase in 150 ul reactions in PNK Buffer (100 U of PNK ; 1X PNK buffer ; 1 mM ATP; 40U RNAseOUT). Reactions were incubated for 30 min at 37°C and heat-inactivated at 75°C for 15 min. DNA was extracted with Phenol/Chloroform, EtOH precipitated and resuspended in 50 μl Tris Buffer pH=8. Samples were heat-denatured again for 5 min and digested with lambda-exonuclease in 100 μl of custom lambda buffer (67 mM glycine-KOH pH= 8.8, 2.5 mM MgCl2, 50 μg/ml BSA) with 2.5 U lambda-exonuclease/μg DNA (Thermo Scientific) and 80U RNAseOUT and incubated overnight at 37°C. Reactions were inactivated for 10 min at 75°C and DNA was purified with MinElute columns (QIAGEN), as indicated by the instruction manual. RNA primers were hydrolyzed by incubating the eluate for 30 min at 37°C in 0.25 N NaOH, followed by neutralization with acetic acid and purified again with MinElute columns.

tag: cell type

tag: experiment type

tag: dna content

tag: s-phase sub-stage

tag: cell-type


title: ZF-DMS3-HA_MORC7-FLAG_antiFLAG_ChIP

tag: antibody

tag: target protein


title: CD11c+ DCs treated with PBS_rep1

description: CD11c+ DCs treated with PBS_rep1


title: DHO1_1B

tag: rhcmv status

tag: Sex

value: F


title: OriSSDS Mouse ESCs Sample 1

description: Cultured Embryonic Stem Cells

source_name: Leading strand capture and single stranded DNA sequencing for mouse cultured ESCs (Sample 1)

tag: positive selection marker

tag: negative selection marker


title: CD11c+ DCs treated with IL-33_rep3

description: CD11c+ DCs treated with IL-33_rep3

tag: treatment

value: treated with IL-33


title: DHO1_6A

source_name: CD8+NKG2A/C+ T cell

tag: animal id

tag: age (year)


title: ZF-DMS3-HA_MORC7-FLAG_antiHA_ChIP

tag: tissue


title: CD11c+ DCs treated with IL-33_rep2

description: CD11c+ DCs treated with IL-33_rep2


title: MeiS_wtLM_S1_2to4C_STRA8_DMRT1.mm10.RT

description: Whole genome sequencing

data_processing:
Library strategy: DNA-Seq
----------ALIGNMENT----------
BWA mem 0.7.12 (Li, 2013; Li and Durbin, 2009) was used to align to the reference genome (mm10 for mouse; hg38 for human).
-------RT BEDGRAPHS-------
WGS from a non-replicating population was used to calibrate the expected GC-biases from different sequencing instruments (Illumina HiSeq 2500, Illumina HiSeq X, Illumina NextSeq 500). Calibration files were generated by examining a subset of perfectly mappable genomic loci. Each locus is a 101 bp region with the following properties:
1. Full mappability of all bases within the 101 bp window. To determine mappability, we generated a fastq file of pseudoreads of the requisite length from the reference genome fasta file. One pseudo-read was generated per bp; all bases were assigned a q-score of 60. Pseudo-reads were mapped to the reference genome using bwa mem 0.7.12 and the coverage at each base in the genome was assessed.
2. The window does not overlap with sequencing gaps (UCSC Gaps track), segmental duplications (UCSC SegDups track), high copy repeats (UCSC repeatmasker track) or otherwise blacklisted genomic regions (Amemiya et al., 2019; Kent et al., 2002).
The GC content (rounded to 1%) and WGS sequencing coverage of each 101 bp window was assessed. The median coverage for all windows of a given GC content was used as a correction factor. These correction factors vary substantially across the different sequencing platforms used. 2% of all qualifying 101 bp autosomal windows were used to calculate the GC correction coefficients. Coverage in each window was corrected using these coefficients.
Replication timing was calculated as the log2 ratio of corrected sequencing coverage at a locus to the genome-wide median (method was adapted from (Koren et al., 2014)). RT-Seq coverage was smoothed in 500 Kb windows with a 50 Kb step. For compatibility with RT-Sim, each 50Kb window is split into five 10Kb bins with equal RT in the RT BEDGRAPH files.

source_name: Whole genome sequencing for RT-Seq in MeiS (wild-type littermate of SPO11)_RT_WGS

growth_protocol: All DNA was harvested from organs.

extract_protocol:
Nuclei from mouse or human testes were prepared as described in (Lam et al., 2019). To purify the populations of interest, a combination of intranuclear markers were used (Table 1, Figure 2). Pure nuclei populations were resuspended in 300 ul of lysis buffer (SDS 1%, 10mM EDTA, 50mM, Tris pH=8) and then sonicated in a Bioruptor UCD200 for 20 min 30 sec ON 30 sec OFF, “high” setting. NaCl was added to a final concentration of 0.2M and the sample incubated overnight at 65C to reverse DNA-protein crosslinks. 5U of Proteinase K (NEB Cat. No. P8107S) was added and the sample further incubated for 1h at 45C. DNA was purified with MinElute columns (QIAGEN), as indicated by the instruction manual.
Whole genome sequencing libraries were prepared from 100 ng of purified DNA using the KAPA Hyper Prep Kit (Cat. No. KK8502) following the manufacturer's instructions.

treatment_protocol: Samples were untreated.

value: Sorted nuclei from testis

value: Whole genome sequencing for RT-Seq

value: Meiotic S-phase


title: SPGI_wtLM_S1_2to4C_DMRT1hi_STRA8.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in SPGI (wild-type littermate of SPO11)_RT_WGS

value: Intermediate stage spermatogonia

value: Strong Dmrt1


title: PolII_ChIP_in_hex

tag: developmental stage

value: anti-PolII Ab817 (Abcam)

value: PolII


title: CD11c+ DCs treated with IL-33_rep1

description: CD11c+ DCs treated with IL-33_rep1


title: PolII_ChIP_in_WT

tag: strain


title: DHO1_3A

tag: fragmentation time (sec)


title: cross-generation_suvh29MORC7-FLAG_ChIP_F3


title: SPGU_SPO11_S1_2to4C_DMRT1_STRA8.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in SPGU(SPO11)_RT_WGS

tag: experiment type

value: Undifferentiated spermatogonia


title: DHO1_2A

value: M


title: ESC_ALL_S1_2to4C_NA_NA.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in ESC(b)_RT_WGS

extract_protocol:
Exponentially growing cells were cross-linked by adding fresh 1% paraformaldehyde solution to the ES media for 10 minutes at room temperature. The cells were washed twice with 1× phosphate-buffered saline and scraped off the plates, pelleted and nuclei were prepared as described in (Lam et al., 2019). Replicating cells were sorted based DNA content.
Whole genome sequencing libraries were prepared from 100 ng of purified DNA using the KAPA Hyper Prep Kit (Cat. No. KK8502) following the manufacturer's instructions.

value: Undifferentiated ES cells

value: Mitotic S-phase

tag: dna content


title: SPGU_ALL_S4_2to4C_DMRT1_STRA8.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in SPGU(b)_RT_WGS

tag: s-phase sub-stage


title: cross-generation_suvh29MORC7-FLAG_ChIP_F2

value: floral tissue

value: anti-FLAG M2 (Sigma)


title: SPGU_ALL_S2_2to4C_DMRT1_STRA8.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in SPGU(a)_RT_WGS

tag: cell-type


title: DHO1_1A

extract_protocol:
Norgen Total RNA Purification Micro Kit
Lexogen QuantSeq 3' mRNA-Seq Library Prep Kit

value: pos


title: MeiS_LATE_S2_2to4C_SCP3yH2AX_NA.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in T4_RT_WGS

value: Weak Sycp3 and weak gH2AX


title: MeiS_MID_S2_2to4C_SCP3yH2AX_NA.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in T3_RT_WGS


title: dms3MORC7-FLAG_ChIPseq_rep-1

value: 5-6 weeks

tag: antibody


title: SPGU_wtLM_S1_2to4C_DMRT1_STRA8.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in SPGU (wild-type littermate of SPO11)_RT_WGS


title: cross-generation_MORC7-FLAG_ChIP

tag: target protein


title: MeiS_EARLY_S2_2to4C_SCP3yH2AX_NA.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in T2a_RT_WGS


title: SPGI_SPO11_S1_2to4C_DMRT1hi_STRA8.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in SPGI(SPO11)_RT_WGS


title: 2C_ALL_S2_2C_NA_NA.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in 2C_MM_RT_WGS

value: Non-replicating


title: SPGI_ALL_S1_2to4C_DMRT1hi_STRA8.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in SPGI(a)_RT_WGS

value: Sorted nuclei from testis


title: DHO1_5A

tag: rhcmv status


title: PIWIL3homo - WGBS_rep2

city: Tokyo

first: Yuka

middle: W.

last: Iwasaki

address: 35 Shinanomachi, Shinjuku-ku,

department: Department of Molecular Biology

institute: Keio University School of Medicine

description: Piwil3KO_FGO_combined_CpG_context_cutoff.bismark.cov.txt.bz2

data_processing:
small-RNA-seq: The adapter sequence and 4nt random sequences at 3’ and 5’ ends were removed cutadapt v1.14. A quality filter of 30 was adopted by removing sequences below this threshold. Reads at the range of 10 ~ 40nt after adapter-removal were used for further analysis. Reads were mapped to the hamster reference genome (hamster.sequel.draft-20200302.arrow.fasta; 3) using Bowtie (v1.1.1), permitting 0 mismatches, and genome-mapped reads were used for further analysis. Genome mapped reads were compared between replicate samples and combined due to their high reproducibility. Reads were further mapped to tRNA, rRNA, sn/snoRNA, and miRNA. The read count mapped to miRNAs were used to normalize small RNA reads obtained from heterozygous and homozygous mutants. Read counts mapped to each unique small RNA sequence were compared between heterozygous and homozygous samples, and those with an 4-fold decrease and identical to previously described PIWI-associated piRNAs were extracted as PIWIL1- or PIWIL3-piRNA.
RNA-seq: Low-quality sequence (a quality value lower than 30) and 3nt random sequence was removed using cutadapt software. Mapping to the golden hamster genome (hamster.sequel.draft-20200302.arrow.fasta; 3) was performed using HISAT2 using default parameters. featureCount v2.01 was used to count reads mapped to genes. featureCounts output was further processed using the TCC-GUI version 2019.02.08 using default parameters. The TCC-GUI uses the TMM normalization method, and edgeR is used for the filtering of differentially expressed genes (DEGs).
Bisulfite-seq: Reads were trimmed to remove low quality bases, and were mapped to the golden hamster genome using Bismark v0.20.0. Methylation data at CG and non-CG sites covered with 10-500 reads were extracted for the downstream analyses.
Genome_build: hamster.sequel.draft-20200302.arrow.fasta (newly sequenced genome available under accession number PRJDB10770 in DDBJ)
Supplementary_files_format_and_content: small-RNA-seq: each unique PIWIL1/PIWIL3-piRNA sequence (see reference for the defenition of PIWIL1/PIWIL3-piRNA) with count of obtained reads in tab separated format.
Supplementary_files_format_and_content: RNA-seq: expression level of genes in each samples calculated by using TCC-GUI (TMM normalization method and edgeR is used in TCC), in csv format.
Supplementary_files_format_and_content: Bisulfite-seq: position of CpG (name of scaffold, start position, and end position), methylation ratio, number of methylated CpG, and number on unmethylated CpG in tab separated format.

organism: Mesocricetus auratus

source_name: PIWIL3homo - WGBS

growth_protocol: Golden hamsters were purchased from Japan SLC and maintained with 14 light and 10 dark cycle. All animal experiments were approved by the Animal Care and Use Committee of the Keio university.

extract_protocol:
Mature females were induced to superovulate by i.p. injection of 40 IU PMSG (ASKA Animal Health) at 1200 to 1400 on the day of conspicuous, postestrus vaginal discharge (day 1 of the estrous cycle). GV oocytes were collected from ovary after 102h PMSG injection. MII oocytes were collected from oviduct after 115h PMSG injection. PN zygotes were collected from oviduct after 91 or 115h PMSG injection followed by mating with male during the night of day4 or 5 (depend on the female estrus cycle).
small-RNA-seq: Total RNAs for small RNA sequence were extracted from MII oocytes of PIWIL1 and PIWIL3 homozygous and heterozygous mutants. The construction of small RNA libraries was performed using NEXTFLEX® Small RNA-Seq Kit v3 (PerkinElmer) with the manufacturer’s instructions.
RNA-seq: Total RNA for RNA-seq were purified from 50 MII oocytes of 2 to 4-month-old golden hamsters using NucleoSpin RNA Plus XS (MACHEREY-NAGEL). RNA-seq libraries were construct using total RNA from MII oocytes and SMART-Seq Stranded Kit (TaKaRa Bio).
Bisulfite-seq: DNA from thirty GV oocytes were spiked with 1% unmethylated lambda phage DNA. Libraries were generated by post bisulfite adaptor tagging method with 8 cycles of library amplification.

treatment_protocol: PIWIL1 and PIWIL3 mutant golden hamster was generated and established using CRISPR/Cas9 system. See reference for details (Hasuwa et al.).

value: GV oocyte

tag: genotype

value: m/m


title: PIWIL3homo - WGBS_rep3


title: DHO1_4A

tag: Sex


title: nrpdnrpeMORC7-FLAG_ChIPseq_rep-1


title: SPGB_ALL_S4_2to4C_STRA8DMRT1_NA.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in SpgB(b)_RT_WGS

value: Whole genome sequencing for RT-Seq

value: A1-A4 and B-type spermatogonia

tag: positive selection marker

value: Stra8 and strong Dmrt1

tag: negative selection marker


title: rpaSSDS_Mouse

description: ChIP-SSDS

data_processing:
Library strategy: RPA-SSDS
Sequencing reads were aligned to the mouse mm10 reference genome using bwa 0.7.12 and the single-stranded DNA pipeline (Brick et al. 2018; Khil et al. 2012). Hotspots were called using the SSDS DSB hotspot calling pipeline (Brick et al. 2018). Hotspot strength was calculated using the method in (Brick et al. 2018). Briefly, each peak is recentred to the midpoint of the Watson and Crick fragment distributions. Signal is estimated as the Crick-strand signal to the left of the origin center + the Watson-strand signal to the right of the center. The background is estimated by counting the remaining fragments at the left and right edges of origins (15%); Watson-strand fragments to the left of the center, Crick-strand fragments to the right. This background is then extrapolated to the entire origin and subtracted from the signal.

source_name: DSB hotspot capture in C57Bl/6 mice using RPA-SSDS in testis

extract_protocol:
Testicular samples were obtained from a single adult mouse. RPA ChIP was performed as described previously for DMC1 (Khil et al., 2012; Brick et al., 2018) with minor modifications.
Sequencing libraries for anti-RPA were prepared following the method described for DMC1-SSDS in (Khil et al., 2012).

value: Testis

value: DSB hotspot mapping using RPA ChIP followed by Single Stranded DNA Sequencing (RPA-SSDS)

value: Anti-RPA32/RPA2 antibody (Abcam, ab10359)


title: PIWIL3homo - WGBS_rep1


title: PIWIL3hetero - WGBS_rep3

description: Piwil3Het_FGO_combined_CpG_context_cutoff.bismark.cov.txt.bz2

source_name: PIWIL3hetero - WGBS

value: w/m


title: nrpeMORC7-FLAG_ChIPseq_rep-2


title: PIWIL3hetero - WGBS_rep1


title: SPGB_ALL_S2_2to4C_STRA8DMRT1hi_NA.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in SpgB(a)_RT_WGS


title: MeiS_EARLY_S3_2to4C_SCP3STRA8_NA.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in T2b_RT_WGS

value: Meiotic S-phase

value: Weak Sycp3 and Stra8


title: nrpeMORC7-FLAG_ChIPseq_rep-1

value: floral tissue

value: anti-FLAG M2 (Sigma)


title: nrpdMORC7-FLAG_ChIPseq_rep-1


title: SPGI_ALL_S4_2to4C_DMRT1hi_STRA8.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in SPGI(b)_RT_WGS

value: Intermediate stage spermatogonia

value: Strong Dmrt1


title: MeiS_VERYEARLY_S1_2to4C_SCP3_NA.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in T1_RT_WGS

value: T1:Very Early

value: Weak Sycp3


title: nrpdMORC7-FLAG_ChIPseq_rep-2

value: 5-6 weeks


title: PIWIL3homo - RNA-seq_rep3

description: PIWIL3_tmm_edger_3_0.1_0.05_TCC_Normalized.csv

source_name: PIWIL3homo - RNA-seq

value: MII oocyte

value: m/m


title: OriSSDS Mouse Whole Testis RNAse Sample 5

data_processing:
Library strategy: Ori-SSDS
----------ALIGNMENT----------
Sequencing reads from Ori-SSDS experiments were aligned to the mouse mm10 genome using bwa 0.7.12 and the single-stranded DNA pipeline (Brick et al. 2018; Khil et al. 2012). Only fragments derived unambiguously from ssDNA (ssDNA_type1) were used in subsequent analyses. ssDNA fragments where either read1 or read2 had mapping quality <30 were discarded. Fragments from the mitochondrial chromosome and fragments in blacklisted regions of the genome were also discarded. Duplicate fragments were subsequently discarded.
----------PEAK CALLING----------
We used the MACS algorithm for peak calling. To assure that data derived from Ori-SSDS fit the MACS peak model, we performed two operations:
1. The strand orientation of fragments was reversed prior to peak calling.
2. The data was passed to MACS as ssDNA fragment BED files (from SSDS pipeline).
Peak calling was performed for each Ori-SSDS sample using MACS 2.1.2 and the following parameters : -g mm -q 0.001 -extsize 2000 --nomodel --nolambda.
For origin calls from individual samples, peaks that did not exhibit the expected Crick-Watson asymmetry were discarded (see paper Figure 1A).
The high-confidence origin set was defined by merging origin calls from wt_Rep1,2,3 and ESC_Rep1. Origin strength was calculated in each sample using a method analogous to that for DMC1-SSDS hotspot mapping(Brick et al. 2018). Briefly, each origin is recentred to the midpoint of the Watson and Crick fragment distributions. Signal is estimated as the Crick-strand signal to the left of the origin center + the Watson-strand signal to the right of the origin center. The background is estimated by counting the remaining fragments at the left and right edges of origins (15%); Watson-strand fragments to the left of the origin center, Crick-strand fragments to the right. This background is then extrapolated to the entire origin and subtracted from the signal. Following this process, origins found in just one sample and narrow origins (<7 Kb) origins that did not display the expected Crick/Watson asymmetry (see paper Figure 1A) in any sample were discarded.
----------BIGWIG files----------
Duplicate fragments were removed from the ssDNA type1 BED file. separate BED files were created from +-strand, --strand and all fragments. Each BED file was converted to a BAM using "bedtools bedtobam" and sorted to match the genome reference fasta file. The bamCoverage tool from deeptools v3.0.1  was used to cenerate a BIGWIG file with the following arguments ( --outFileFormat bigwig --binSize 150 -p max --ignoreForNormalization chrX chrM chrY --normalizeUsing RPKM).

source_name: Leading strand capture and single stranded DNA sequencing for mouse whole testis. RNAse treatment before lambda exonuclease. Experiment performed in parallel with whole testis Sample 5.

extract_protocol:
Mice were euthanized and testes were retrieved. The tunica albuginea was removed and both testes were suspended in 10 ml of DNAzol (Thermo-Fisher catalog number 10503027) and transferred to a Dounce homogenizer. Tissue and cells were disrupted with 5-7 strokes of a loose-fitting pestle. The DNA was precipitated with 5 ml of EtOH and then spooled with a pipette tip and rinsed gently twice with EtOH 75% by sequential transfer into 15 ml tubes. DNA was air-dried for 5 min and then resuspended in 500 μl of 1X TEN buffer (Tris 10mM pH=8; EDTA 1mM; NaCl 100mM) + 80 U of RNAseOUT (40 U/μl) at 4C for at least 24h.
DNA was denatured at 95°C for 5 min, chilled on ice for 5 min and size-fractionated on 5–30% (w/v) linear sucrose gradients in TEN buffer in a Beckman Ultracentrifuge SW45 rotor for 18 h at 26,000 g at 20°C. 500 ul fractions were collected from the top of the gradient, precipitated by adding 1 ml of EtOH 100 % and 50 μl of NaOAc 3M pH 5.2 and resuspended in 25 μl Tris Buffer pH=8. Fractions were analyzed in a denaturing (50 mM NaOH, 1 mM EDTA) 1.2% agarose gel and the ones containing ssDNA ranging in size from 800 to 2000 nt were pooled. RNA primers were hydrolyzed by incubating the eluate for 30 min at 37°C in 0.25 N NaOH, followed by neutralization with acetic acid. Then, samples were heat denatured and spun through a Chromaspin TE-1000 size exclusion column (Clontech). SNS were heat-denatured for 5 min at 95°C and chilled on ice for 5 min and DNA is phosphorylated by Polynucleotide Kinase in 150 ul reactions in PNK Buffer (100 U of PNK ; 1X PNK buffer ; 1 mM ATP; 40U RNAseOUT). Reactions were incubated for 30 min at 37°C and heat-inactivated at 75°C for 15 min. DNA was extracted with Phenol/Chloroform, EtOH precipitated and resuspended in 50 μl Tris Buffer pH=8. Samples were heat-denatured again for 5 min and digested with lambda-exonuclease in 100 μl of custom lambda buffer (67 mM glycine-KOH pH= 8.8, 2.5 mM MgCl2, 50 μg/ml BSA) with 2.5 U lambda-exonuclease/μg DNA (Thermo Scientific) and 80U RNAseOUT and incubated overnight at 37°C. Reactions were inactivated for 10 min at 75°C and DNA was purified with MinElute columns (QIAGEN), as indicated by the instruction manual. RNA primers were hydrolyzed by incubating the eluate for 30 min at 37°C in 0.25 N NaOH, followed by neutralization with acetic acid and purified again with MinElute columns

value: Whole testis

value: Origin of replication mapping using leading strand capture and Single Stranded DNA Sequencing (Ori-SSDS)


title: nrpdnrpeMORC7-FLAG_ChIPseq_rep-2


title: OriSSDS Mouse Whole Testis Sample 5

source_name: Leading strand capture and single stranded DNA sequencing for mouse whole testis (Sample 5)

extract_protocol:
Mice were euthanized and testes were retrieved. The tunica albuginea was removed and both testes were suspended in 10 ml of DNAzol (Thermo-Fisher catalog number 10503027) and transferred to a Dounce homogenizer. Tissue and cells were disrupted with 5-7 strokes of a loose-fitting pestle. The DNA was precipitated with 5 ml of EtOH and then spooled with a pipette tip and rinsed gently twice with EtOH 75% by sequential transfer into 15 ml tubes. DNA was air-dried for 5 min and then resuspended in 500 μl of 1X TEN buffer (Tris 10mM pH=8; EDTA 1mM; NaCl 100mM) + 80 U of RNAseOUT (40 U/μl) at 4C for at least 24h.
DNA was denatured at 95°C for 5 min, chilled on ice for 5 min and size-fractionated on 5–30% (w/v) linear sucrose gradients in TEN buffer in a Beckman Ultracentrifuge SW45 rotor for 18 h at 26,000 g at 20°C. 500 ul fractions were collected from the top of the gradient, precipitated by adding 1 ml of EtOH 100 % and 50 μl of NaOAc 3M pH 5.2 and resuspended in 25 μl Tris Buffer pH=8. Fractions were analyzed in a denaturing (50 mM NaOH, 1 mM EDTA) 1.2% agarose gel and the ones containing ssDNA ranging in size from 800 to 2000 nt were pooled. To match conditions used for the RNA hydrolysis control, samples were incubated for 30 min at 37°C in Tris Buffer pH=8. Then, samples were heat denatured and spun through a Chromaspin TE-1000 size exclusion column (Clontech). SNS were heat-denatured for 5 min at 95°C and chilled on ice for 5 min and DNA is phosphorylated by Polynucleotide Kinase in 150 ul reactions in PNK Buffer (100 U of PNK ; 1X PNK buffer ; 1 mM ATP; 40U RNAseOUT). Reactions were incubated for 30 min at 37°C and heat-inactivated at 75°C for 15 min. DNA was extracted with Phenol/Chloroform, EtOH precipitated and resuspended in 50 μl Tris Buffer pH=8. Samples were heat-denatured again for 5 min and digested with lambda-exonuclease in 100 μl of custom lambda buffer (67 mM glycine-KOH pH= 8.8, 2.5 mM MgCl2, 50 μg/ml BSA) with 2.5 U lambda-exonuclease/μg DNA (Thermo Scientific) and 80U RNAseOUT and incubated overnight at 37°C. Reactions were inactivated for 10 min at 75°C and DNA was purified with MinElute columns (QIAGEN), as indicated by the instruction manual. RNA primers were hydrolyzed by incubating the eluate for 30 min at 37°C in 0.25 N NaOH, followed by neutralization with acetic acid and purified again with MinElute columns


title: OriSSDS Mouse Whole Testis RNAse Sample 4

source_name: Leading strand capture and single stranded DNA sequencing for mouse whole testis. RNAse treatment before lambda exonuclease. Experiment performed in parallel with whole testis Sample 4.


title: MeiS_BxCMID_R1_2to4C_SCP3_NA.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in MeiS(BxC)M_RT_WGS

value: Sorted nuclei from testis

value: C57BL6J X CAST/EIJ F1

value: Whole genome sequencing for RT-Seq

value: Mid

value: Weak Sycp3 and weak gH2AX


title: PIWIL3homo - RNA-seq_rep2


title: OriSSDS Mouse Whole Testis Sample 4

source_name: Leading strand capture and single stranded DNA sequencing for mouse whole testis (Sample 4)


title: MeiS_SPO11_S1_2to4C_STRA8_DMRT1.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in MeiS(SPO11)_RT_WGS

value: Meiotic S-phase


title: WT-FLAG_ChIPseq_rep-2

value: floral tissue


title: WT-FLAG_ChIPseq_rep-1

value: anti-FLAG M2 (Sigma)


title: OriSSDS Mouse Whole Testis Sample 3

source_name: Leading strand capture and single stranded DNA sequencing for mouse whole testis (Sample 3)

extract_protocol:
Mice were euthanized and testes were retrieved. The tunica albuginea was removed and both testes were suspended in 10 ml of DNAzol (Thermo-Fisher catalog number 10503027) and transferred to a Dounce homogenizer. Tissue and cells were disrupted with 5-7 strokes of a loose-fitting pestle. The DNA was precipitated with 5 ml of EtOH and then spooled with a pipette tip and rinsed gently twice with EtOH 75% by sequential transfer into 15 ml tubes. DNA was air-dried for 5 min and then resuspended in 500 μl of 1X TEN buffer (Tris 10mM pH=8; EDTA 1mM; NaCl 100mM) + 80 U of RNAseOUT (40 U/μl) at 4C for at least 24h.
DNA was denatured at 95°C for 5 min, chilled on ice for 5 min and size-fractionated on 5–30% (w/v) linear sucrose gradients in TEN buffer in a Beckman Ultracentrifuge SW45 rotor for 18 h at 26,000 g at 20°C. 500 ul fractions were collected from the top of the gradient, precipitated by adding 1 ml of EtOH 100 % and 50 μl of NaOAc 3M pH 5.2 and resuspended in 25 μl Tris Buffer pH=8. Fractions were analyzed in a denaturing (50 mM NaOH, 1 mM EDTA) 1.2% agarose gel and the ones containing ssDNA ranging in size from 800 to 2000 nt were pooled. SNS were heat-denatured for 5 min at 95°C and chilled on ice for 5 min and DNA is phosphorylated by Polynucleotide Kinase in 150 ul reactions in PNK Buffer (100 U of PNK ; 1X PNK buffer ; 1 mM ATP; 40U RNAseOUT). Reactions were incubated for 30 min at 37°C and heat-inactivated at 75°C for 15 min. DNA was extracted with Phenol/Chloroform, EtOH precipitated and resuspended in 50 μl Tris Buffer pH=8. Samples were heat-denatured again for 5 min and digested with lambda-exonuclease in 100 μl of custom lambda buffer (67 mM glycine-KOH pH= 8.8, 2.5 mM MgCl2, 50 μg/ml BSA) with 2.5 U lambda-exonuclease/μg DNA (Thermo Scientific) and 80U RNAseOUT and incubated overnight at 37°C. Reactions were inactivated for 10 min at 75°C and DNA was purified with MinElute columns (QIAGEN), as indicated by the instruction manual. RNA primers were hydrolyzed by incubating the eluate for 30 min at 37°C in 0.25 N NaOH, followed by neutralization with acetic acid and purified again with MinElute columns.


title: PIWIL3homo - RNA-seq_rep1


title: PIWIL3hetero - RNA-seq_rep3

source_name: PIWIL3hetero - RNA-seq

value: w/m


title: MeiS_ALL_S4_2to4C_STRA8_DMRT1.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in MeiS(b)_RT_WGS


title: MORC7-FLAG_ChIPseq_rep-3


title: OriSSDS Mouse Whole Testis Sample 2

source_name: Leading strand capture and single stranded DNA sequencing for mouse whole testis (Sample 2)


title: MeiS_ALL_S5_2to4C_STRA8_DMRT1.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in MeiS(c)_RT_WGS


title: MORC7-FLAG_ChIPseq_rep-2


title: PIWIL3hetero - RNA-seq_rep2


title: PIWIL3hetero - WGBS_rep2

source_name: PIWIL3hetero - WGBS

value: GV oocyte


title: MORC4-FLAG_ChIPseq_rep-2


title: MeiS_ALL_S3_2to4C_STRA8_DMRT1.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in MeiS(a)_RT_WGS


title: Lep_ALL_S1_4C_SCP3STRA8_NA.mm10.RT

description: Whole genome sequencing

source_name: Whole genome sequencing for RT-Seq in 4C_LEP_RT_WGS

value: Leptonema

value: Strong Sycp3 and Stra8


title: PIWIL3hetero - RNA-seq_rep1


title: 2C_ALL_MMC_2C_NA_NA

description:
Whole genome sequencing
RTseqCorrectionFiles.tar.gz

source_name: Whole genome sequencing for RT-Seq in 2C_MMC_RT_WGS

value: Non-replicating


title: PIWIL1homo - RNA-seq_rep3

description: PIWIL1_tmm_edger_3_0.1_0.05_TCC_Normalized.csv

source_name: PIWIL1homo - RNA-seq

value: MII oocyte


title: MORC4-FLAG_ChIPseq_rep-1

value: 5-6 weeks


title: Mesenchymal single cells from C57BL/6 mice for control

first: Shiyuan

middle: (Cynthia)

last: Chen

department: Computational Biology

institute: Stowers Institute for Medical Research

description:
getting single cell without treatment
ControlMCA

data_processing:
Raw sequencing data were processed using 10X Genomics Cell Ranger pipeline (version 2.1.1). Reads were demultiplexed into Fastq file format using cellranger mkfastq. Genome index was built by cellranger mkref using cavefish genome astMex1, ensembl 87 gene model. Data were aligned by STAR aligner and cell counts tables were generated using cellranger count function with default parameters.
Genome_build: mm10
Supplementary_files_format_and_content: cellranger gene count h5 matrix

source_name: C57BL/6 mice Intestine

growth_protocol: C57BL/6, BALB/c, R26-LSL-EGFP, ApcMin, Bmi1-CreER, LGR5-GFP were obtained from The Jackson Laboratory. Male and female mice up to 200 days old were used for all studies. All mice were housed in the AAALAC-accredited animal facility at the Stowers Institute for Medical Research (SIMR) and handled according to SIMR and National Institutes of Health guidelines. All procedures involving experimental animals were approved by the Institutional Animal Care and Use Committee (IACUC) of SIMR under protocol 2019-070.

extract_protocol:
Open intestine longitudinally. Wash intestine 5 times with ice-cold HBSS w/o Ca2+Mg2+. Add 37.6 ml HBSS w/o Ca2+Mg2+ to 50 ml Falcon tube. Add 2.4 ml of 0.5M EDTA (10 mM). Add in the small pieces of intestine (2-3 cm pieces) with the final volume 40 ml, invert tube to mix; incubate on ice for 20 min, wash 5 times with 40 ml cold HBSS w/o Ca2+Mg2+ and incubate on ice for 20 min. Shake vigorously for 4 min. Keep the supernatant in a new tube, and add 40ml cold HBSS w/o Ca2+Mg2+. Shake vigorously for 4 min. Repeat until getting 3 tubes of supernatant. (preparation of EP) Use 40 µm cell strainer to filter supernatant. Centrifuge (Jouan CR422) the tubes at approximately 300g for 5 min at 4°C.  Remove the supernatant and add 2 ml of TryPLE Express (containing 0.05 mM NAC and 10 µM Y-27632) per sample. Incubate for 4 min in a 37°C water bath with gently shaking in hands or stirring with a P100 pipette occasionally. Place on ice for 1 min, gently shaking. Transfer dissociated samples into 40 ml ice cold ADMEM/F12 washing medium (containing 1% L-GlytaMax, 1% HEPES, 1% Penicillin/Streptomycin, 0.05 mM NAC and 10 µM Y-27632). Shake the 50-ml tube for about 30 sec to enhance the dissociation and use 70 µm cell strainer to filter supernatant. Spin down at 500g for 5 min at 4°C. Re-suspend pellet in 3ml of ice cold ADMEM/F12 washing medium, use 20 µm syringe filter to separate any remaining mass and then get the epithelial single cell. (preparation of MC) Add 10 ml ice-cold HBSS with Ca2+Mg2+. Pre-heat Liberase enzyme solution 370C for 10min. (Prepare Enzyme solution: 10 ml HBSS with Ca2+Mg2+ , 300 ul Liberase in 50 ml Falcon tube) Transfer mesenchymal intestinal preparation to pre-heated Liberase enzyme solution. Incubate 370C for 20’ with occasional gentle shaking (2X/min) Cool 50 ml tube on ice for 3 min. Add 200ul DNase I (2,000 U/ml). Shake vigorously (2x/second) to dissociate mesenchymal tissue. Transfer samples to MACS commercial tubes. Use Intestine_01 program for about a minute in MACS dissociator. Transfer dissociated tissue to 50 ml Falcon tube; add 1 ml 0.5M EDTA; mix gently by shaking. Add 30 ml HBSS with Ca2+Mg2+ + 3% FBS (900 ul) immediately; mix gently by inverting. Resuspend a few times using a 30ml syringe + 16G needle. Filter tissue through 40µm cell strainer (DB) directly into a new 50 ml tube. Pre-heat 4ml of lysis buffer in a warm bath. Centrifuge at 1600 rpm (~ 600g) 40C 7 min. to pellet cells. Add 2 ml of Hemolytic lysis buffer and keep the tube in a water bath for less than 2 min. Add up to 45ml of MACS Rinsing solution + Y27632 inhibitor to stop lysis. Filter with 70um cell strainer (DB) after lysis. Centrifuge at 500xg for 5 min at 4 degree, and resuspend with HBSS without Ca2+Mg2+ + 0.4%  BSA.
Dissociated cells, having been sorted in HBSS without Ca2+Mg2+ + 0.4%  BSA, were assessed for concentration and viability via a Nexelom Cellometer Auto T4.  Cells, having been deemed to be at least 60% viable, were loaded on a Chromium Single Cell Controller (10x Genomics, Pleasanton, CA), based on live cell concentration. Libraries were prepared using the Chromium Single Cell 3' Library & Gel Bead Kit v2 (10x Genomics) according to manufacturer’s directions.  Resulting short fragment libraries were checked for quality and quantity using an Agilent 2100 Bioanalyzer and Invitrogen Qubit Fluorometer.  Libraries were pooled in groups, at equal molar concentrations.  With cells captured estimated at ~420-19,000 cells per sample, libraries were sequenced to a depth necessary to achieve 8,500-175,000 mean reads per cell - ~40-340M reads each - on an Illumina HiSeq 2500 instrument using Rapid SBS v2 chemistry with the following paired read lengths: 26 bp Read1, 8 bp I7 Index and 98 bp Read2.
10X scRNA-seq

treatment_protocol: APCMin/+ mice were treated with one-time 5gy and 5-FU, and harvest each intestine from mice different time points.

tag: cell type

value: Intestinal Mesenchymal single cell from C57BL/6 mice

tag: tumor type

value: Normal wild type

tag: treatment

value: no treatment

tag: treatment time

value: untreated


title: PIWIL1homo - RNA-seq_rep2

tag: tissue

value: m/m


title: OriSSDS Mouse Sperm Sample 1

source_name: Leading strand capture and single stranded DNA sequencing for mouse sperm (Sample 1)

extract_protocol:
Sperm was retrieved from cauda and caput epididymides from 3 adult mice. The mix of somatic cells and sperm was resuspended in 0.1% SDS, 0.5% Triton X to selectively lyse somatic cells. Sperm was recovered by centrifugation and lysed in 6 M guanidinium, 30 mM sodium citrate (pH 7.0), 0.5% Sarkosyl, 0.20 mg/ml proteinase K and 0.3 M Beta-mercaptoethanol, and incubated at 55°C for 1h (ref).  The DNA was precipitated with 2 volumes of isopropyl alcohol and then spooled with a pipette tip and rinsed gently twice with EtOH 75% by sequential transfer into 15 ml tubes. DNA was air-dried for 5 min and then resuspended in 500 μl of 1X TEN buffer (Tris 10mM pH=8; EDTA 1mM; NaCl 100mM) + 80 U of RNAseOUT (Thermo-Fisher catalog number 10777019) (40 U/μl) at 4C for at least 24h
DNA was denatured at 95°C for 5 min, chilled on ice for 5 min and size-fractionated on 5–30% (w/v) linear sucrose gradients in TEN buffer in a Beckman Ultracentrifuge SW45 rotor for 18 h at 26,000 g at 20°C. 500 ul fractions were collected from the top of the gradient, precipitated by adding 1 ml of EtOH 100 % and 50 μl of NaOAc 3M pH 5.2 and resuspended in 25 μl Tris Buffer pH=8. Fractions were analyzed in a denaturing (50 mM NaOH, 1 mM EDTA) 1.2% agarose gel and the ones containing ssDNA ranging in size from 800 to 2000 nt were pooled. SNS were heat-denatured for 5 min at 95°C and chilled on ice for 5 min and DNA is phosphorylated by Polynucleotide Kinase in 150 ul reactions in PNK Buffer (100 U of PNK ; 1X PNK buffer ; 1 mM ATP; 40U RNAseOUT). Reactions were incubated for 30 min at 37°C and heat-inactivated at 75°C for 15 min. DNA was extracted with Phenol/Chloroform, EtOH precipitated and resuspended in 50 μl Tris Buffer pH=8. Samples were heat-denatured again for 5 min and digested with lambda-exonuclease in 100 μl of custom lambda buffer (67 mM glycine-KOH pH= 8.8, 2.5 mM MgCl2, 50 μg/ml BSA) with 2.5 U lambda-exonuclease/μg DNA (Thermo Scientific) and 80U RNAseOUT and incubated overnight at 37°C. Reactions were inactivated for 10 min at 75°C and DNA was purified with MinElute columns (QIAGEN), as indicated by the instruction manual. RNA primers were hydrolyzed by incubating the eluate for 30 min at 37°C in 0.25 N NaOH, followed by neutralization with acetic acid and purified again with MinElute columns.

value: Sperm

tag: strain

value: Origin of replication mapping using leading strand capture and Single Stranded DNA Sequencing (Ori-SSDS)


title: Mesenchymal single cells from APCmin mice before treatment

description:
getting single cell without treatment
APCminMC

source_name: APCmin/+ mice Intestine

value: Intestinal Mesenchymal single cell from APCmin/+ mice

value: Adenoma


title: Mesenchymal single cells from APCmin mice before treatment b

description:
getting single cell without treatment
APCminMCb


title: PIWIL1hetero - RNA-seq_rep3

source_name: PIWIL1hetero - RNA-seq


title: PIWIL1homo - RNA-seq_rep1


title: Epithelial single cells from APCmin mice before treatment

description:
getting single cell without treatment
APCminEP

value: Intestinal Epithelial single cell from APCmin/+ mice


title: Epithelial single cells from APCmin mice before treatment b

description:
getting single cell without treatment
APCminEPb


title: OriSSDS Mouse Whole Testis Sample 1

source_name: Leading strand capture and single stranded DNA sequencing for mouse whole testis (Sample 1)

value: Whole testis

tag: experiment type


title: 48 hour Mesenchymal single cells from APCmin mice after treatment

description:
getting single cell after 48h with treatment
48hMC

value: Irradiation:5Gy, Fluorouracil (5-FU) injection


title: Naïve Neutrophil

city: Yokohama

first: Hidehiro

last: FUKUYAMA

state: Kanagawa

address: 1-7-22, Suehirocho, Tsurumi-ku

department: IMS

institute: RIKEN

data_processing:
Read alignment: TopHat2 v2.0.8 with default settings
expression level: Cufflinks v2.1.1 with default settings
FPKM (Fragments per Kirobase per Million reads) were calculated.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample ...

source_name: Bone marrow

extract_protocol:
Bone marrow cells were prepared from mice 48 h after systemic LPS injection and sorted using a cell sorter. Sorted cells were lysed in TRIzol LS reagent (Thermo Fisher Scientific) and total RNA was extracted using RNeasy Mini or Micro Kit (QIAGEN), a FavorPrep Total RNA Extraction Column (Favorgen, Taiwan) according to the manufacturer’s protocol.
DNA library preparation for RNA-Seq analysis using a SureSelect Strand-Specific RNA Library Prep Kit for Illumina Multiplexed Sequencing (Agilent Technologies).

treatment_protocol: LPS (100 ug/mouse) was intravenously injected into Ym1-Venus mice.

tag: age

value: 7 weeks

tag: lps treatment

value: No treatment

value: Bone marrow

value: Neutrophil (Ly6G+)


title: PIWIL1hetero - RNA-seq_rep2

value: MII oocyte

value: w/m


title: Naïve Monocyte

value: Ly6Chi monocyte (Ly6C+ CD115+ Venus-)


title: 48 hour Epithelial single cells from APCmin mice after treatment

description:
getting single cell after 48h with treatment
48hEP


title: Epithelial single cells from C57BL/6 mice for control

description:
getting single cell without treatment
ControlEPA

value: Intestinal Epithelial single cell from C57BL/6 mice

value: Normal wild type

value: no treatment


title: PIWIL1hetero - RNA-seq_rep1


title: mice 1 of non-treadmill exercise LD2

city: Guilin City

first: Cao

last: Zhen

address: 1 Zhiyuan Road, Lingui, Guilin, Guangxi 541004, P.R. China

country: China

institute: Guilin medical university

description: miRNAs expression data from femur of mice

hyb_protocol: The CustomArray™ microarray was pre-hybridized in nuclease-free water at 65°C for 10 min, and then loaded the microarray onto the rotisserie in the hybridization oven and incubated at 37°C for 60 min with gentle rotation.The hybridization solution was prepared with labeled microRNA target and denatured the hybridization solution at 95°C for 3 min, and then cooled for 20 s on ice. Then the microarray was loaded with the hybridization solution and incubated at 37°C for 16 h with gentle rotation.

scan_protocol: Removed the microarray from the hybridization solution and washed the microarray by using the wash solution to remove nonspecific hybridization. Then covered the semiconductor microarray surface with the imaging solution and loaded the microarray into the scanner to scan. Imaging was performed using a laser scanner GenePix4000B

data_processing: The data was analyzed by Guangzhou Ribo Bio Co., Ltd. normalization method is quantile.

source_name: Femur

label_protocol: Part of the total RNA was purified with the mirVana MiRNA Isolation kit (Ambion; Thermo Fisher Scientific, Inc.) and labeled with Cyanine 3 using the ULS™ mirVana miRNA ArrayLabeling Kit (Ambion; Thermo Fisher Scientific, Inc.).

growth_protocol: 6 mice had free access to food and water under a relative humidity of 40-70˚C, temperature of 22-25˚C and 12-h light/dark cycle.

extract_protocol: On average, 3 femur of treadmill exercise mice and  3 femur of non-treadmill exercise mice were prepared for the extraction of total RNA, RNA quantity and purity were assessed by Guangzhou RiboBio Co., Ltd.

treatment_protocol: The treadmill training group (treadmill speed, 13 m/min; slope, 9°; training for 40 min per day at the same time each day, 6 days/week) and the control group (no treadmill training)

value: Femur

value: 16 weeks old

value: Male

tag: exercise

value: non-treadmill exercise


title: mice 1 of non-treadmill exercise LD3


title: mice 3 of treadmill exercise LS3

value: treadmill exercise


title: mice 1 of treadmill exercise LS1


title: scRNA-seq_GO

city: Daejeon

first: Jaeeun

last: Jung

address: 125 Gwahak-ro, Youseong-gu

country: South Korea

institute: KRIBB

data_processing:
Digital gene expression matrices for all samples were demultiplexed and aligned to hg38 using CellRanger v3
Digital gene expression matrices were generated using CellRanger v3
Genome_build: hg38
Supplementary_files_format_and_content: digital gene expression matrix

source_name: Organoid

extract_protocol:
Illumina TruSeq kit for paired-end reads, poly-A selection, non-strand-specific
10X Chromium Single Cell 3' (V3), manufacturer's protocol

value: iPSC-derived organoid


title: Venus(+)Ly6Chi Monocyte

value: 48h after LPS

value: Ly6Chi monocyte (Ly6C+ CD115+ Venus+)


title: 96 hour Mesenchymal single cells from APCmin mice after treatment

description:
getting single cell after 96h with treatment
96hMC

value: Intestinal Mesenchymal single cell from APCmin/+ mice

value: Adenoma

value: Irradiation:5Gy, Fluorouracil (5-FU) injection


title: mice 1 of non-treadmill exercise LD1


title: 96 hour Epithelial single cells from APCmin mice after treatment

description:
getting single cell after 96h with treatment
96hEP

value: Intestinal Epithelial single cell from APCmin/+ mice


title: scRNA-seq_LBO


title: MLO-Y4 osteocytes of mechanical loading WS2

description: miRNAs expression data from MLO-Y4 osteocytes

source_name: MLO-Y4 osteocytes

growth_protocol: A mouse MLO-Y4 osteocyte cell line  was cultured in dishes withα-MEM medium,containing 10% FBS and 1% penicillin. Then the cells were transferred to mechanical loading dishes that were reformed from cell culture dishes

extract_protocol: Total RNA extraction and miR enrichment procedures were performed using the Trizol method and an mirVana miR Isolation kit (Ambion Life Technologies, Carlsbad,CA, USA).

treatment_protocol: The MLO-Y4 cells were stimulated with mechanical tensile strain of 2500με at 0.5 Hz for 8 h by a four-point bending device

tag: cell

value: MLO-Y4 osteocytes

tag: sample group

value: mechanical loading


title: LPS Neutrophil

value: 7 weeks

value: Bone marrow

value: Neutrophil (Ly6G+)


title: Primed WT rep2

data_processing:
Raw reads were trimmed with cutadapt to remove low quality bases
Trimmed reads were aligned to hg38 using STAR_2.5.4b with gencode v27 annotation
Aligned reads were quantified using featureCounts 1.6.3 from the subreads package
Genome_build: hg38
Supplementary_files_format_and_content: gene counts text file

source_name: hPSC

growth_protocol: H9 primed hESCs were cultured in mTeSR Plus on Matrigel hESC-Qualified Matrix coated wells and H9 naïve hESCs were maintained on mitomycin C inactivated MEFs feeder cells in PXGL media consisted of N2B27 supplemented with 1 μM  PD0325901, 2 μM XAV939, 2 μM Gö6983, and 20 ng/mL human LIF. All naïve hESC experiments were conducted in 5% O2, 5% CO2. For re-priming, naïve cells were single-cell dissociated using TrypLE Express and seeded 5 x 10^5 cells per Matrigel-coated well in mTeSR Plus supplemented with 10uM Y-27632. After two days, Y-27632 was withdrawn. The cells were harvested for RNA-seq analysis five days after transfer to mTeSR Plus media. Primed and re-primed cultures were conducted in 20% O2 and 5% CO2.

extract_protocol:
Total RNA was isolated using the E.Z.N.A. total RNA kit I.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: hPSC

tag: iPSc source

value: Human Embryonic Stem cells

tag: passage

tag: genotype/variation

value: WT


title: MLO-Y4 osteocytes of mechanical loading WS3


title: 8 hour Mesenchymal single cells from APCmin mice after treatment

description:
getting single cell after 8h with treatment
8hMC


title: Primed WT rep3


title: 8 hour Epithelial single cells from APCmin mice after treatment

description:
getting single cell after 8h with treatment
8hEP


title: 3 hour Mesenchymal single cells from APCmin mice after treatment

description:
getting single cell after 3h with treatment
3hMC


title: MLO-Y4 osteocytes of mechanical loading WS1


title: Re-Priming BRG1 KO rep3

value: KO


title: 3 hour Epithelial single cells from APCmin mice after treatment

description:
getting single cell after 3h with treatment
3hEP


title: MLO-Y4 osteocytes of non-mechanical loading WD3

value: non-mechanical exercise


title: Re-Priming BRG1 KO rep2


title: MLO-Y4 osteocytes of non-mechanical loading WD2


title: Re-Priming BRG1 KO rep1


title: 24 hour Epithelial single cells from APCmin mice after treatment

description:
getting single cell after 24h with treatment
24hEP


title: Testis - wt - smRNA-seq_rep3

data_processing:
small-RNA-seq: The adapter sequence and 4nt random sequences at 3’ and 5’ ends were removed cutadapt v1.14. A quality filter of 30 was adopted by removing sequences below this threshold. Reads at the range of 10 ~ 40nt after adapter-removal were used for further analysis. Reads were mapped to the hamster reference genome (hamster.sequel.draft-20200302.arrow.fasta; 3) using Bowtie (v1.1.1), permitting 0 mismatches, and genome-mapped reads were used for further analysis. Genome mapped reads were compared between replicate samples and combined due to their high reproducibility. Reads were further mapped to tRNA, rRNA, sn/snoRNA, and miRNA. The read count mapped to miRNAs were used to normalize small RNA reads obtained from heterozygous and homozygous mutants. Read counts mapped to each unique small RNA sequence were compared between heterozygous and homozygous samples, and those with an 4-fold decrease and identical to previously described PIWI-associated piRNAs were extracted as PIWIL1- or PIWIL3-piRNA.
Genome_build: hamster.sequel.draft-20200302.arrow.fasta (newly sequenced genome available under accession number PRJDB10770 in DDBJ)
Supplementary_files_format_and_content: small-RNA-seq: each unique PIWIL1/PIWIL3-piRNA sequence (see reference for the defenition of PIWIL1/PIWIL3-piRNA) with count of obtained reads in tab separated format.

source_name: Testis - wt - smRNA-seq

extract_protocol:
Mature females were induced to superovulate by i.p. injection of 40 IU PMSG (ASKA Animal Health) at 1200 to 1400 on the day of conspicuous, postestrus vaginal discharge (day 1 of the estrous cycle). GV oocytes were collected from ovary after 102h PMSG injection. MII oocytes were collected from oviduct after 115h PMSG injection. PN zygotes were collected from oviduct after 91 or 115h PMSG injection followed by mating with male during the night of day4 or 5 (depend on the female estrus cycle).
small-RNA-seq: Total RNAs for small RNA sequence were extracted from MII oocytes of PIWIL1 and PIWIL3 homozygous and heterozygous mutants or testis of PIWIL1 homozygous mutant and wild type. The construction of small RNA libraries was performed using NEXTFLEX® Small RNA-Seq Kit v3 (PerkinElmer) with the manufacturer’s instructions.

value: Testis

tag: genotype

value: w/w


title: 24 hour Mesenchymal single cells from APCmin mice after treatment

description:
getting single cell after 24h with treatment
24hMC


title: Venus(-)Ly6Chi Monocyte

value: 48h after LPS

value: Ly6Chi monocyte (Ly6C+ CD115+ Venus-)


title: Re-Priming BRG1 HET

value: HET


title: 0 hour Mesenchymal single cells from APCmin mice after treatment

description:
getting single cell after 0h with treatment
0hMC

value: Intestinal Mesenchymal single cell from APCmin/+ mice

value: Adenoma


title: Re-Priming BRG1 WT


title: Testis - PIWIL1homo - smRNA-seq_rep3

source_name: Testis - PIWIL1homo - smRNA-seq

value: m/m


title: 0 hour Epithelial single cells from APCmin mice after treatment

description:
getting single cell after 0h with treatment
0hEP

value: Intestinal Epithelial single cell from APCmin/+ mice


title: Testis - wt - smRNA-seq_rep2


title: MLO-Y4 osteocytes of non-mechanical loading WD1

value: MLO-Y4 osteocytes


title: Naive BRM KO rep2


title: mice 2 of treadmill exercise LS2

value: Femur

value: 16 weeks old

tag: Sex

value: Male

value: treadmill exercise


title: Testis - PIWIL1homo - smRNA-seq_rep1


title: CTRL-2AN_S6

city: Charleston

first: Jeremy

last: Barth

state: SC

address: 173 Ashley Ave, BSB 627

institute: MUSC

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads (fastq files) were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence
Secondary analysis was carried out using Partek Flow. Alignment was conducted with TopHat2 using mouse genome (mm10) and the default option set
Aligned reads were quantified to annotation model (Partek E/M) using mm10 and RefSeq transcripts 83 (2017-11-01) with the settings: a) strict paired-end compatibility, b) require junction reads to match introns, c) min read overlap with feature 100% of read length, d) min reads 10
Gene counts were analyzed using DESeq2 to determine normalized expression measures and signficant differences (p-adjusted) for all pairwise comparisons among the postnatal samples
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text file of normalized counts and comparison metrics

source_name: auditory nerve

extract_protocol:
Microdissections were performed to isolate the auditory nerve from mouse cochlear structures. Two cochleae from each mouse were pooled for individual samples. Total RNA was purified from each sample using the miRNeasy Mini Kit per the manufacturer’s instructions. Quality of each total RNA isolation was assessed using the Agilent 2100 Bioanalyzer and low-quality samples showing degradation or contamination were excluded.
RNA libraries were prepared for sequencing using standard Illumina protocols using Illumina's TruSeq RNA Library Prep Kit

value: auditory nerve

value: adult


title: Naive BRM KO rep1

tag: cell type

value: hPSC

value: Human Embryonic Stem cells

value: KO


title: Naive BRG1 KO rep2


title: CTRL-1-AN_S1


title: Naive BRG1 KO rep1


title: P21-AN3_S27

value: postnatal day 21


title: PIWIL3homo - smRNA-seq_rep2

source_name: PIWIL3homo - smRNA-seq

value: MII oocyte


title: Primed WT rep1

value: WT


title: Naive WT rep2


title: PIWIL3homo - smRNA-seq_rep1


title: P21-AN1_S17


title: Testis - PIWIL1homo - smRNA-seq_rep2

value: Testis


title: PIWIL3homo - smRNA-seq_rep3


title: Naive WT rep1


title: Testis - wt - smRNA-seq_rep1

value: w/w


title: P14-AN2_S23

value: postnatal day 14


title: P14-AN1_S18


title: P14-AN3_S28


title: P7-AN6_S16

value: postnatal day 7


title: PIWIL1homo - smRNA-seq_rep3

source_name: PIWIL1homo - smRNA-seq

value: m/m


title: PIWIL3hetero - smRNA-seq_rep1

source_name: PIWIL3hetero - smRNA-seq

value: w/m


title: PIWIL3hetero - smRNA-seq_rep3


title: PIWIL1homo - smRNA-seq_rep1


title: KLF4_naive_rep1

data_processing:
Library strategy: CUT&Tag
Raw reads were processed by AIAP v1.1 (https://www.biorxiv.org/content/10.1101/686808v1). Briefly, reads were quality trimmed by cutadapt. Trimmed reads were aligned by bwa. Aligned reads were quanitfied using methylQA density and peaks called with macs2. Signal density files normalized to 10 million reads were also generated.
Genome_build: hg38
Supplementary_files_format_and_content: BigWig density file and peaks call bed file

growth_protocol: WIBR3 naïve hESCs were cultured on mitomycin C inactivated MEFs feeder cells in 5i/L/A media consisted of N2B27 supplemented with 1 μM PD0325901, 1 μM IM-12, 0.5 μM SB590885, 1 μM WH4-023, 10 μM Y-27632, 20 ng/mL hLIF, and 10 ng/mL Activin A and WIBR3 primed hESCs were maintained on mitomycin C inactivated MEFs feeder cells in primed hESC medium containing bFGF.

extract_protocol:
200,000 cells per sample replicate were washed in Wash Buffer [1 mL 1 M HEPES pH 7.5 (Sigma Aldrich, H3375), 1.5 mL 5 M NaCl (Sigma Aldrich, S5150), 12.5 μL 2 M Spermidine (Sigma Aldrich, S2501), 1 Roche Complete Protease Inhibitor EDTA-Free tablet (Sigma Aldrich, 5056489001), and bring the final volume to 50 mL with dH2O], then immobilized on 10 ul of Concanavalin A-coated beads (Bangs Laboratories). Cells were cleared on a magnetic rack, then permeabilized with Dig-wash buffer [20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine and 1x protease inhibitor cocktail containing 0.05 % Digitonin]. The cells were then incubated with primary antibody KLF4 (R&D, AF3640, 1:100) at 4°C overnight. The primary antibody was cleared on a magnetic rack. Rabbit anti-Goat IgG secondary antibody was diluted 1:100 in Dig-wash buffer and incubated at RT for an hour. Cells were cleared on a magnetic rack and washed with 1 mL of Dig-wash buffer. A 1:200 diluted of pA-Tn5 adapter complex was prepared in Dig-300 Buffer [20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM Spermidine and 1x protease inhibitor cocktail containing 0.05 % Digitonin]. Cells were cleared on a magnetic rack and incubated by adding 100 ul of pA-Tn5 at RT for 1 hour. Cells were washed with 1 mL of Dig-300 buffer, resuspended in 300 ul of Tagmentation buffer [10 mM MgCl2 in Dig-300 Buffer], and incubated at 37℃ for 1 hour. 10 ul of 0.5 M EDTA, 3 ul of 10% SDS and 2.5 ul of 20 mg/mL Proteinase K was added to each reaction to stop the tagmentation at 55℃ for an hour. DNA was purified using phenol/chloroform/isoamyl alcohol (PCI) extraction followed by chloroform extraction and precipitated with glycogen and ethanol. DNA was pelleted with a high-speed spin at 4℃, washed, air dried for 5 min and resuspended in 50 ul of double-distilled water (Kaya-Okur et al., 2019).
The DNA was PCR amplified using i5 and i7 indexing primers, and cleaned up with AMPure XP beads (Beckman Coulter, A63880). Agilent 4200 TapeStation analysis was performed for size distribution of libraries and as desired aiming for a final concentration, libraries were mixed to achieve equal representation. Paired-end Illumina sequencing on the barcoded libraries was used by following the manufacturer’s instruction.


title: CTRL-3AN_S11


title: P7-AN5_S12


title: PIWIL3hetero - smRNA-seq_rep2


title: KLF4_primed


title: shYTHDF2_Control_STAMP [10X]

city: La Jolla

first: Gene

last: Yeo

address: 2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab

institute: UCSD

description:
cells dissociated from solid tumor
shYTHDF2_Control_STAMP_S8_L004

data_processing:
Alignment, cell barcode processing, umis processing, abundance measurements: cellranger count (version 3.0.2)
MD tags were added to alignments with samtools calmd --threads 15 -rb possorted_genome_bam.bam refdata-cellranger-hg19-3.0.0/fasta/genome.fa > possorted_genome_bam_MD.bam
Reads were split based on the CB:Z tag, resulting in one BAM file per barcode.
Edits were called on each "split-BAM" file using SAILOR (http://github.com/yeolab/sailor) using default parameters and dbSNP (v147) to remove known SNPs.
Coverage was generated from filtered BAM files (intermediates from SAILOR) for each cell barcode, using the following commands from BedTools (v2.27.1). :     bedtools genomecov -split -strand - -g refdata-cellranger-hg19-3.0.0/star/chrNameLength.txt -bg -ibam fwd.bam > fwd.bg  bedtools sort -I fwd.bg > fwd.sorted.bg   bedGraphToBigWig fwd.sorted.bg refdata-cellranger-hg19-3.0.0/star/chrNameLength.txt fwd.sorted.bw    bedtools genomecov -split -strand + -g refdata-cellranger-hg19-3.0.0/star/chrNameLength.txt -bg -ibam rev.bam > rev.bg  bedtools sort -I rev.bg > rev.sorted.bg   bedGraphToBigWig rev.sorted.bg refdata-cellranger-hg19-3.0.0/star/chrNameLength.txt rev.sorted.bw
For each barcode, edit sites were annotated across exons (Gencode v19) and the cumulative C>T depth across each gene was divided by the cumulative depth across C's as obtained from the previous step.
Genome_build: hg19
Supplementary_files_format_and_content: 10X: Edit fractions across barcodes with detectable edits were merged to obtain processed STAMP_edits.txt files for each sample.

growth_protocol: MDA-MB-231-LM2 cells were cultured in DMEM + 10% FBS. Cells were split every 3-4 days. For 10X experiments cells were subcutaneously injected in the flank of mice and grown for 6 weeks before tumor dissociation and 10X library preparation.

extract_protocol:
10X Chromium Single Cell 3' polyA v3 Chemistry
10X Chromium Single Cell 3' polyA and feature capture v3 Chemistry

treatment_protocol: Mice were maintained on 5% sucrose water with 2mg/mL Doxycycline for 3 days before tumor extraction and dissociation.

value: Doxycycline

tag: cell culture

value: DMEM + 10% FBS


title: shYTHDF2_RPS2_STAMP [10X]

description:
cells dissociated from solid tumor
shYTHDF2_RPS2_STAMP_S6_L004

growth_protocol: MDA-MB-231-LM2 cells were cultured in DMEM + 10% FBS. Cells were split every 3-4 days. Cells were subcutaneously injected in the flank of mice and grown for 6 weeks before tumor dissociation and 10X library preparation.

tag: treatment


title: PIWIL1hetero - smRNA-seq_rep3

source_name: PIWIL1hetero - smRNA-seq


title: P21-AN2_S22


title: WT_Control_STAMP [10X]

description:
cells dissociated from solid tumor
WT_Control_STAMP_S1_L004

data_processing:
Alignment, cell barcode processing, umis processing, abundance measurements: cellranger count (version 3.0.2)
MD tags were added to alignments with samtools calmd --threads 15 -rb possorted_genome_bam.bam refdata-cellranger-hg19-3.0.0/fasta/genome.fa > possorted_genome_bam_MD.bam
Reads were split based on the CB:Z tag, resulting in one BAM file per barcode.
Edits were called on each "split-BAM" file using SAILOR (http://github.com/yeolab/sailor) using default parameters and dbSNP (v147) to remove known SNPs.
Coverage was generated from filtered BAM files (intermediates from SAILOR) for each cell barcode, using the following commands from BedTools (v2.27.1). :     bedtools genomecov -split -strand - -g refdata-cellranger-hg19-3.0.0/star/chrNameLength.txt -bg -ibam fwd.bam > fwd.bg  bedtools sort -I fwd.bg > fwd.sorted.bg   bedGraphToBigWig fwd.sorted.bg refdata-cellranger-hg19-3.0.0/star/chrNameLength.txt fwd.sorted.bw    bedtools genomecov -split -strand + -g refdata-cellranger-hg19-3.0.0/star/chrNameLength.txt -bg -ibam rev.bam > rev.bg  bedtools sort -I rev.bg > rev.sorted.bg   bedGraphToBigWig rev.sorted.bg refdata-cellranger-hg19-3.0.0/star/chrNameLength.txt rev.sorted.bw
For each barcode, edit sites were annotated across exons (Gencode v19) and the cumulative C>T depth across each gene was divided by the cumulative depth across C's as obtained from the previous step.
Genome_build: hg19
Supplementary_files_format_and_content: Edit fractions across barcodes with detectable edits were merged to obtain processed STAMP_edits.txt files for each sample.


title: KLF4_naive_rep2


title: Naive BRG1 HET

value: HET


title: P3-AN2_S13

value: auditory nerve

value: postnatal day 3


title: PIWIL1hetero - smRNA-seq_rep1


title: PIWIL1homo - smRNA-seq_rep2


title: 34150

city: Duarte

first: Hsun

middle: Teresa

last: Ku

address: 1500 E. Duarte Road

department: Translational Research and Cellular Therapeutics

institute: City of Hope

data_processing:
bcl2fastq v2.20 (loaded by cellranger mkfastq (3.0.1)) software used for basecalling.
reads were aligned to refdata-cellranger-hg19-1.2.0 genome assembly using cellranger v3.0.1
Counting data of reference gene expression levels were generated by cellranger v3.0.1 (automatic identification of chemistry properties)
Genome_build: hg19
Supplementary_files_format_and_content: Tab separated values; MatrixMarket

source_name: Adult human exocrine pancreas

extract_protocol:
Single cell suspension was obtained from fresh human fetal pancreas by collagenase P digestion. 10,000 single cells were captured on a 10xGenomics Chromium controller using a Chromium single cell 3' library & Gel Bead kit V3 kit
Reverse transcription was performed in emulsion droplet with 10xGEM beads, full length of cDNA was amplified and 3' expression library was constructed with Tn5 transposase based method (with 10xGenomics chromium single cell 3' Library Constraction Kit V3).

tag: developmental stage

value: Adult

value: Pancreas


title: P3-AN1_S9

tag: tissue


title: P7_AN4_S12