title: NMFs from healthy BALB/C mice repetition 1

description: Library name: balb1


title: spermatozoa, proven-fertile, 8

status: Public on Jan 16 2025

type: SRA

city: Providence

first: Daniel

middle: James

last: Spade

state: RI

address: Box G-E5

department: Pathology and Laboratory Medicine

country: USA

institute: Brown University

description:
no duplicate
8

library_source: transcriptomic

data_processing:
Raw sequence data (.bcl files) generated by the Illumina HiSeq Control Software (HCS) was converted into fastq files and de-multiplexed using Illumina bcl2fastq 2.20 software. One mis-match was allowed for index sequence identification.
Sequence reads were trimmed to remove adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36.
The trimmed reads were mapped to the Homo sapiens GRCh38 reference genome available on ENSEMBL using the STAR aligner v.2.5.2b.
Unique gene hit counts were calculated by using feature Counts from the Subread package v.1.5.2. Only unique reads that fell within exon regions were counted.
Three subjects in the infertility group were sequenced in two samples, and these technical duplicates are combined. This yields a final count matrix of dimension 57500 x 67 where each row represents a gene with its unique ENSEMBL ID, and each column represents a subject.
Before conducting DA analysis, we first filtered genes with low counts among all samples (sum over all sequencing samples below 700; roughly 10 counts per sample). For the 27,809 genes that passed the filtering, we applied median normalization to account for differences in sequencing depth among samples. The normalized counts are log 2 transformed after adding one pseudo count.
Assembly: GRCh38
Supplementary files format and content: .fastq.gz files contain raw counts for each sample
Supplementary files format and content: log2_norm_count_collapsed.csv is a matrix of log2-transformed counts for all samples

molecule: polyA RNA

organism: Homo sapiens

source_name: ejaculate

extract_protocol:
Sperm were isolated from the ejaculate by a washing and somatic cell lysis procedure, followed by isolation with the miRvana kit, as described in Bianchi et al. (2018. doi: 10.1111/andr.12471).
RNA libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA). mRNAs were selected using oligo-dT beads, fragmented for 15 minutes at 94 °C, followed by first strand and second strand cDNA synthesis, followed by adaptor ligation, indexing, and PCR enrichment.

tag: tissue

value: ejaculate

tag: cell type

value: spermatozoa

tag: population

value: proven-fertile


title: Rbp4,F,Saline,48hrs,rep3,b2

city: Berkeley

first: Andrea

middle: M

last: Gomez

state: CA

address: 163 Weill Hall

department: Molecular and Cell Biology

institute: University of California, Berkeley

description: Library name: AG-2677

data_processing:
Data quality assessment and sequence read primer trimming were performed by FastQC v0.11.9 and Cutadapt 3.5, respectively. We considered trim_galore version 0.6.7 with --phred33 parameter. The data was then aligned to a mouse reference genome build (Mus musculus; GRCm39: RefSeq assembly accession = GCF_000001635.27) using RSEM v1.3.1. Bowtie2 version 2.4.4 aligner index was created using rsem-prepare-reference --bowtie2 command and a gene-level read count matrix was generated using rsem-calculate-expression --bowtie2 command. Gene-level counts of each sample were combined using rsem-generate-data-matrix command.
For alternative splicing analysis, we first applied the same procedures for assessing data quality and sequence read primer trimming. The reads were then aligned to a mouse reference genome build (Mus musculus; GRCm39) using STAR 2.7.10a.
Assembly: Mus musculus; GRCm39
Supplementary files format and content: tab-delimited text file includes raw counts for each sample
Supplementary files format and content: tab-delimited text file include raw counts for all samples combined

organism: Mus musculus

source_name: mouse prefrontal cortex

extract_protocol:
RNA purification from RiboTag mice were modified from previously published protocols (see methods for details). RNA purification was performed using a RNeasy Mini kit following the manufacturer’s instructions.
Samples with RNA integrity number (RIN) values >7 were used for library prep. 50 ng of RNA was used for library preparation with TruSeq stranded RNA library prep kit with poly-A enrichment. The libraries were sequenced 150bp paired-end on a NovaSeq 6000 platform for DOI samples, and X Plus for psilocybin samples, at 200 million reads per sample.

treatment_protocol: DOI (2mg/kg), psilocybin (2mg/kg) or saline were injected intraperitoneally (IP).

value: mouse prefrontal cortex

tag: cre line

tag: Sex

value: female

tag: replicate

tag: time

tag: treatment

value: Saline

tag: batch


title: NK.meninges.24h.DT#6

city: Boston

first: CBDM

last: Lab

state: MA

address: 77 Avenue Louis Pasteur

department: Microbiology and Immunobiology

institute: Harvard Medical School

description: 6-weeks-old Foxp3DTR+ male littermates were intraperiotoneally injected (i.p.)  with diptheria toxin. The meninges were harvested 24 hours after the injection and Tconv (CD45+ TCRb+ CD4+ CD8- Foxp3-) cells and NK cells (CD45+ Lin (CD19, TCRb, CD11c, gdTCR, CD64)- NK1.1 +) were isolated and sorted for sequencing.

data_processing:
Reads were mapped to the mouse reference genome (mm10) with STAR 2.7.3a
Non-uniquely mapped reads were discarded.
The uniquely mapped reads were assigned to genes using featureCounts with GENCODE annotation (vM25)
Assembly: mm10
Supplementary files format and content: tab-delimited text files include raw read counts for each Sample

molecule: total RNA

source_name: Meninges

extract_protocol:
For freshly-isolated samples, 500-1000 cells were double-sorted into 5ul TCL buffer (Qiagen) containing 1% 2-mercaptoethanol (Sigma-Aldrich). For whole-tissue RNA-seq, the tissues were harvested and lysed in QIAzol followed by RNA isolation according to the manufacturer's protocol and 2 ng RNA were resuspended in 5 microliters of TCL buffer containing 1% 2-mercaptoethanol.
Library construction was performed according to the standard Immunological Genome Project protocols (https://www.immgen.org/img/Protocols/ImmGenULI_RNAseq_methods.pdf).

value: Meninges

value: NK cell

tag: genotype

value: diptheria toxin


title: Rbp4,F,Saline,1week,rep5,b2

description: Library name: AG-2686


title: spermatozoa, infertility, 783L

description:
duplicate: 775
775L

value: infertility


title: Rbp4,F,DOI,48hrs,rep3,b2

description: Library name: AG-2676

value: DOI


title: Rbp4,M,DOI,48hrs,rep4,b2

description: Library name: AG-2692

value: male


title: Rbp4,F,Saline,48hrs,rep4,b2

description: Library name: AG-2688


title: NK.meninges.24h.DT#5


title: RMS_Snord101_Ctrl_B

city: Houston

first: Eric

last: Van Nostrand

state: TX

address: 1 Baylor Plaza Rm T334

department: Therapeutic Innovation Center

institute: Baylor College of Medicine

description: RiboMeth-seq from mouse induced neurons treated with non-targeting ASO, biological replicate 2

data_processing:
Adapter trimming was performed using either trim_galore or cutadapt.
Mapping was performed using bowtie2 with --end-to-end -k 1 options.
Mapped bam files were converted to bed using bedtools bamtobed then 5' and 3' end counting was performed using "awk '{print $2}' RMS_${sample}.bed | sort | uniq -c | awk '{print $2, $1}' | sort -n > RMS_${sample}.5count.txt" and "awk '{print $3}' RMS_${sample}.bed | sort | uniq -c | awk '{print $2, $1}' | sort -n > {.}.3count.txt"
RMS meth score was calculated considering weighted 5' or 5'+3' counts of neighboring 12 nucleotides as described in Birkedal et al. 2014.
Assembly: Reads were mapped to 45S rRNA (NR_046233.2) and U2 snRNA (NR_004414.1).
Supplementary files format and content: Tab delimited text file containing end counts (5' only or 5' and 3') and the calculated RMS score at each nucleotide position in 45S rRNA or U2 snRNA for each of the replicate RMS libraries. Mean RMS scores are also provided.
Library strategy: RiboMeth-seq

source_name: ESC

growth_protocol: mESCs were maintained on plates pre-coated with 0.2% gelatin in DMEM (Thermo) supplemented with 10 mM HEPES (Thermo), 0.11 mM β-mercaptoethanol (Sigma), 1X nonessential amino acids (Corning), 2 mM L-glutamine (Corning), 15% fetal bovine serum (Cytivia), and 1000 U/mL leukemia inhibitory factor (LIF; Sigma).

extract_protocol:
Total RNA was extracted using Trizol.
150 to 250 ng of DNase-treated RNA was hydrolyzed in 50 mM sodium bicarbonate buffer (pH 9.2) for 1 to 12 minutes. The hydrolysis reaction was quenched in 1 mL EtOH containing 10 uL 3 M NaOAc, pH 5.2 (Thermo) and 15 ug Glycoblue coprecipitant (Thermo) and submerged in liquid nitrogen. Hydrolyzed RNA was then precipitated and washed in 80% EtOH. The intended fragment size of around 50-100 bp was verified on the TapeStation 4200 system (Agilent). RNA was dephosphorylated by Antarctic Phosphatase (New England Biolabs) for 30 minutes at 37°C, and end-repaired by T4 PNK (New England Biolabs) in the presence of 1 mM ATP (New England Biolabs) for 1 hour at 37°C. End-repaired RNA was purified using RNA Clean & Concentrator-5 columns (Zymo). Illumina libraries were then prepared using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® kit (New England Biolabs) and purified on NucleoSpin® Gel and PCR Clean-up columns (Takara Bio).

treatment_protocol: For ASO experiments, mESC were first subject to in vitro neuron differentiated followed by free uptake of ASO, as follows. mESC were cultured for 24 hours on gelatin-coated plates in neuron differentiation media [BrainPhys neuronal medium (Stem Cell Technologies) supplemented with 1X NeuroCult SM1 (Stem Cell Technologies), 1X N2 supplement (Thermo), 20 ng/mL brain-derived neurotrophic factor (Stem Cell Technologies)] containing 1 μg/mL doxycycline (Sigma) to induce Ngn2 expression. Then, cells were re-plated onto 0.1% PEI coated dishes in neuron differentiation media containing 1 ug/mL doxycycline (Sigma). Within 24 hours of re-plating, 5-10-5 MOE gapmer ASOs (IDT) were added to the culture media at a final concentration of 10 uM to allow for free uptake by the neurons. RNA was extracted 72 to 96 hours after addition of the ASO using TRIzol (Thermo). The ASO sequences were Snord101 ASO 5’-GGGTATCCGACAATTCAAGT-3’ and Snord89 ASO 5’-TGACTCGCTTCAGGAGGTTT-3’.

tag: cell line

value: ESC

value: iNeuron

value: non-targeting ASO


title: RMS_Snord101_KD_B

description: RiboMeth-seq from mouse induced neurons treated with Snord101-targeting ASO, biological replicate 2

value: Snord101 ASO


title: PV,F,DOI,48hrs,rep1,b2

description: Library name: AG-2627

value: PV


title: NK.meninges.24h.DT#4


title: spermatozoa, infertility, 775L

description:
duplicate: 783
775L

value: ejaculate


title: RMS_Snord101_Ctrl_A

description: RiboMeth-seq from mouse induced neurons treated with non-targeting ASO, biological replicate 1


title: NK.meninges.24h.PBS#3

value: PBS


title: Cux2ERT,M,Saline,1week,rep3,b2

description: Library name: AG-2622


title: spermatozoa, infertility, 772L

description:
duplicate: 765
765L

value: spermatozoa


title: MBP_3

city: Beijing

first: Haiyue

last: Yu

address: Yuanmingyuanxilu No.2

country: China

institute: Chinese Academy of Agricultural Sciences

description: Library name: MBP_3

data_processing:
Raw reads were firstly subjected to quality control using FastQC (v.0.20.1). The filtered reads were mapped to the Zea mays reference genome (https://download.maizegdb.org/Zm-B73-REFERENCE-GRAMENE-4.0/Zm-B73-REFERENCE-GRAMENE-4.0.fa.gz) using BWA mem (v0.7.12). The MACS2 (version 2.1.0) peak calling software to identify regions of IP enrichment over background. The RIP target were identified based on the default parameters (P-value<0.05, and Fold change>2).
Assembly: B73
Supplementary files format and content: BigWig

organism: Zea mays

source_name: leaves

extract_protocol:
RNA was extracted using Trizol reagent
The fragment distribution and concentration of RNAs（after immunoprecipitation） were detected using an Agilent 2100 bioanalyzer (Agilent) and simpliNano spectrophotometer (GE Healthcare). Then, immunoprecipitated RNA or Input was used for library construction with NEBNext® Ultra™ II RNA Library Prep Kit for Illumina®(New England Biolabs, E7770). Library quality was assessed on the Agilent Bioanalyzer 2100 system. The libraries were sequenced on an Illumina Novaseq platform with a paired-end read length of 150 bp according to the standard protocols.

value: leaves


title: Cux2ERT,M,DOI,1week,rep4,b2

description: Library name: AG-2621

value: mouse prefrontal cortex


title: spermatozoa, infertility, 770L

description:
no duplicate
770L


title: NK.meninges.24h.PBS#2


title: spermatozoa, infertility, 768L

description:
no duplicate
768L


title: MBP_2

description: Library name: MBP_2


title: Cux2ERT,M,DOI,1week,rep3,b2

description: Library name: AG-2618


title: NK.meninges.24h.PBS#1


title: RMS_Snord101_KD_A

description: RiboMeth-seq from mouse induced neurons treated with Snord101-targeting ASO, biological replicate 1


title: spermatozoa, infertility, 784L

description:
no duplicate
784L


title: MBP_1

description: Library name: MBP_1


title: spermatozoa, infertility, 767L

description:
no duplicate
767L


title: Treg.meninges.24h.DT#6

value: Treg cell


title: PV,F,Saline,1month,rep5,b2

description: Library name: AG-2476

value: female

value: Saline


title: EtEC81_3

description: Library name: EtEC81_3


title: Treg.meninges.24h.DT#5

value: Meninges

value: diptheria toxin


title: Cux2ERT,F,DOI,1month,rep2,b2

description: Library name: AG-2450

value: DOI


title: spermatozoa, infertility, 766L

description:
no duplicate
766L

value: infertility


title: EtEC81_2

description: Library name: EtEC81_2


title: spermatozoa, infertility, 765L

description:
duplicate: 772
765L


title: Cux2ERT,M,DOI,1month,rep1,b2

description: Library name: AG-2449

value: male


title: EtEC81_1

description: Library name: EtEC81_1


title: PV,M,DOI,1month,rep4,b2

description: Library name: AG-2413


title: Treg.meninges.24h.PBS#3


title: AGSEBV_sh_GPX4_3

city: Qingdao

first: Duo

last: Shi

address: Qingdao University, 308 Ningxia Road, Qingdao

department: School of Basic Medicine

institute: Qingdao University

description: Library name: AGSEBV_sh_GPX4_3

data_processing:
Illumina Casava1.7 software used for basecalling.
Assembly: GRCh38
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample

source_name: AGS-EBV

extract_protocol:
Total RNA was extracted using the TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol.
The libraries were constructed using VAHTS Universal V6 RNA-seq Library Prep Kit according to the manufacturer’s instructions

value: AGS-EBV

value: EBV positive gastric cancer cells

value: GPX4 knockdown group


title: spermatozoa, infertility, 759L

description:
no duplicate
759L


title: PV,M,Saline,1month,rep4,b2

description: Library name: AG-2411

value: PV


title: Treg.meninges.24h.PBS#2


title: AGSEBV_sh_GPX4_2

description: Library name: AGSEBV_sh_GPX4_2


title: spermatozoa, infertility, 755L

description:
no duplicate
755L

value: ejaculate


title: PV,M,Saline,1month,rep3,b2

description: Library name: AG-2410


title: Treg.meninges.24h.PBS#1

value: PBS


title: spermatozoa, infertility, 754L

description:
no duplicate
754L

value: spermatozoa


title: PV,F,DOI,1month,rep3,b2

description: Library name: AG-2407


title: AGSEBV_sh_GPX4_1

description: Library name: AGSEBV_sh_GPX4_1


title: TH&HTH.DTR-#2

source_name: Thalamus + Hypothalamus

extract_protocol: For freshly-isolated samples, 500-1000 cells were double-sorted into 5ul TCL buffer (Qiagen) containing 1% 2-mercaptoethanol (Sigma-Aldrich). For whole-tissue RNA-seq  , the tissues were harvested and lysed in QIAzol followed by RNA isolation according to the manufacturer's protocol and 2 ng RNA were resuspended in 5 microliters of TCL buffer containing 1% 2-mercaptoethanol. Library construction and sequencing and data processing were performed according to the standard Immunological Genome Project protocols (https://www.immgen.org/img/Protocols/ImmGenULI_RNAseq_methods.pdf).

value: Thalamus + Hypothalamus

value: Whole tissue

value: Diphtheria toxin


title: TH&HTH.DTR-#3


title: spermatozoa, infertility, 752L

description:
no duplicate
752L


title: AGSEBV_sh_NC_2

description: Library name: AGSEBV_sh_NC_2

value: Control group


title: AGSEBV_sh_NC_3

description: Library name: AGSEBV_sh_NC_3


title: spermatozoa, infertility, 751L

description:
no duplicate
751L


title: PV,M,DOI,1week,rep3,b2

description: Library name: AG-2402

value: mouse prefrontal cortex


title: spermatozoa, infertility, 764L

description:
no duplicate
764L


title: TH&HTH.DTR+#3


title: TH&HTH.DTR-#1


title: PV,M,DOI,1week,rep4,b2

description: Library name: AG-2406


title: Treg.meninges.24h.DT#4

value: Meninges

value: Treg cell


title: WT_52x_2rep

city: Austin

first: Svetlana

last: Panina

address: 101 East 21st St.

department: Molecular Biosciences

institute: The University of Texas at Austin

description: Library name: WT_52x_2rep

data_processing:
Clean single-end data were trimmed with cutadapt v.4.6, using sequence ‘AGATCGGAAGAGCACACGTCT’ as 3’-adapter (-a) and sequence ‘GTTCAGAGTTCTACAGTCCGACGATC’ as 5’-adapter (-g). Only reads with minimal length of 17 bp were retained after trimming.
Next, trimmed reads were mapped onto pre-indexed human reference genome (hg38) using bowtie2 v.2.3.5.1 with the following parameters -N 0 -k 5 --ignore-quals.
Quantification was performed using featureCounts v.1.5.0 with parameters  ‘-t exon -O -s 1 -M - T 4’ and pre-filtered gencode hg38 .gtf file containing genomic coordinates of small RNAs: miRNAs, snoRNAs, snRNAs, and miscRNAs.
Assembly: hg38
Supplementary files format and content: tab-delimited text files include counts for each Sample

growth_protocol: Human embryonic kidney cells (HEK293T) were routinely cultured in Dulbecco’s-modified Eagle’s media (DMEM, Sigma-Aldrich, St. Louis, MO) supplemented with 10% Opti-Gold fetal bovine serum, FBS (GenDEPOT, Katy, TX) and 1% HyClone penicillin and streptomycin (P/S) mix (Cytiva, Marlborough, MA) at 37 °C in a humidified 5% CO2 atmosphere.

extract_protocol:
Total RNA was isolated from HEK293T cells (at least ~106 cells per sample) using TRI reagent (Sigma-Aldrich) and DirectZol RNA Miniprep kit (Zymo Research, Irvine, CA, #R2050/#R2070).
Small RNA-Seq (single-end strategy 50bp) libraries were prepared at Novogene Co. according to the manufacturer’s instructions for the Illumina platform (Illumina, San Diego, CA).

treatment_protocol: To generate 52X T4A and 52X WT CTD HEK293T cells, transient transfection of 1.5 μg of either plasmid (per 1 well of 6-well plate) was performed using PEI (polyethylenimine) reagent (6.6 μl per 1 well). Next day after transfection, cells were selected with α-amanitin (2.5 μg/ml, Sigma-Aldrich, #A2263) for 72 hours with daily drug replenishment based on preliminary time-course experiment that had shown that untransfected cells are eliminated by this time-point.

value: Human embryonic kidney

value: WT

value: a-amanitin


title: spermatozoa, infertility, 744L

description:
no duplicate
744L

value: infertility


title: PV,F,Saline,1week,rep4,b2

description: Library name: AG-2398

value: female

value: Saline


title: TH&HTH.DTR+#1


title: spermatozoa, infertility, 746L

description:
no duplicate
746L


title: Cux2ERT,M,Saline,48hrs,rep2,b2

description: Library name: AG-2276

value: male


title: T4A_52x_2rep

description: Library name: T4A_52x_2rep


title: spermatozoa, infertility, 743L

description:
duplicate: 738
738L

value: ejaculate


title: Cux2ERT,M,DOI,48hrs,rep2,b2

description: Library name: AG-2275

value: DOI


title: PV,F,DOI,1week,rep2,b2

description: Library name: AG-2399

value: PV


title: AGSEBV_sh_NC_1

description: Library name: AGSEBV_sh_NC_1

value: AGS-EBV

value: EBV positive gastric cancer cells


title: Cerebelum.DTR-#3

source_name: Cerebellum

value: Cerebellum

value: Whole tissue

value: Diphtheria toxin


title: T4A_52x_1rep

description: Library name: T4A_52x_1rep


title: spermatozoa, infertility, 740L

description:
no duplicate
740L

value: spermatozoa


title: Cerebelum.DTR-#2


title: Cerebelum.DTR-#1


title: E3

city: Trento

first: Erik

last: Dassi

state: TN

address: Via Sommarive, 9

department: CIBIO

country: Italy

institute: University of Trento

description:
sh1_sin003_3
UTN_SIN_E3_BIAGIOLI_S13

data_processing:
Quality control was performed with TrimGalore, setting Q30 as minimum quality, removing adapters and reads with >1 N calls.
Reads were aligned to the hg38 human genome with STAR 2.5.2b, using the GeneCounts quantification mode option
Assembly: hg38
Supplementary files format and content: tab-separated values, raw by-gene counts

source_name: hiNPCs

growth_protocol: Cells were cultured on poly-L-ornithine hydrobromide (20 µg/mL, Sigma)/laminin (3 µg/mL, Life Technologies) - coated plates in hiNPC medium (70% v/v DMEM (Life Technologies) completed with 30% v/v HAM F12 (Euroclone), 2% v/v B27 (Life Technologies), 1% v/v Penicillin-Streptomycin solution (Life Technologies) and 1% v/v L-Glutamine (Corning) and supplemented with EGF (20 ng/mL, Sigma), bFGF (20 ng/mL, R&D) and Heparin (5 µg/mL, Sigma). Semi-confluent monolayers of hiNPCs were maintained in 5% CO2, 37°C humidified incubator.

extract_protocol:
Total RNA was extracted using TRIZOL reagent protocol and then treated with DNase.
For the library preparation strategy, 100 ng of total RNA was used as input with the Illumina Stranded mRNA Prep kit, followed by 13 PCR cycles. Sequencing was performed on the NovaSeq6000 platform using an SP-200 Flow Cell, with paired-end reads of 100 cycles each (100x2) and a loading concentration of 0.56 nM

treatment_protocol: hiNPC and patient derived fibroblasts were plated at 60% confluency in medium without antibiotics. Cells were treated with 1.5 RTU of lentiviral vectors in the presence 4 ng/ml of polybrene (Sigma). The medium was changed with complete medium supplemented with 1% v/v Penicillin-Streptomycin solution (Life Technologies) after incubation ON at 37°C and cells were collected after 48h of incubation

value: hiNPCs

value: biol rep 3


title: E4

description:
sh1_sin003_4
UTN_SIN_E4_BIAGIOLI_S8

value: biol rep 4


title: TH&HTH.DTR+#2

value: Thalamus + Hypothalamus


title: Cux2ERT,F,Saline,48hrs,rep1,b2

description: Library name: AG-2273

value: mouse prefrontal cortex


title: Cerebelum.DTR+#3


title: spermatozoa, infertility, 738L

description:
duplicate: 743
738L

value: infertility


title: spermatozoa, infertility, 750L

description:
no duplicate
750L


title: Rbp4,M,Saline,48hrs,rep2,b2

description: Library name: AG-2253

value: Saline


title: Cux2ERT,F,DOI,48hrs,rep1,b2

description: Library name: AG-2271

value: female


title: spermatozoa, infertility, 735L

description:
no duplicate
735L

value: ejaculate


title: Rbp4,F,Saline,48hrs,rep1,b2

description: Library name: AG-2250


title: E2

description:
sh1_sin003_2
UTN_SIN_E2_BIAGIOLI_S9

value: biol rep 2


title: Cerebelum.DTR+#2


title: WT_52x_1rep

description: Library name: WT_52x_1rep

value: Human embryonic kidney

value: WT

value: a-amanitin


title: spermatozoa, infertility, 729L

description:
no duplicate
729L


title: spermatozoa, infertility, 734L

description:
no duplicate
734L


title: D3

description:
sh1_sin001_3
UTN_SIN_D3_BIAGIOLI_S3


title: E1

description:
sh1_sin003_1
UTN_SIN_E1_BIAGIOLI_S20

value: biol rep 1


title: Cerebelum.DTR+#1

value: Cerebellum

value: Whole tissue


title: Rbp4,F,DOI,48hrs,rep1,b2

description: Library name: AG-2247

value: DOI


title: Rbp4,F,DOI,48hrs,rep2,b2

description: Library name: AG-2248


title: Cortex.DTR-#3

source_name: Cortex

value: Cortex

value: Diphtheria toxin


title: D2

description:
sh1_sin001_2
UTN_SIN_D2_BIAGIOLI_S6

value: hiNPCs


title: Cortex.DTR-#2


title: PV,M,Saline,1week,rep2,b2

description: Library name: AG-2244

value: PV

value: male


title: PV,M,Saline,1week,rep3,b2

description: Library name: AG-2246

value: mouse prefrontal cortex


title: spermatozoa, infertility, 718L

description:
no duplicate
718L

value: spermatozoa

value: infertility


title: Cortex.DTR-#1


title: PV,M,DOI,1week,rep1,b2

description: Library name: AG-2243


title: spermatozoa, infertility, 711L

description:
no duplicate
711L

value: ejaculate


title: D1

description:
sh1_sin001_1
UTN_SIN_D1_BIAGIOLI_S10


title: spermatozoa, infertility, 730L

description:
no duplicate
730L


title: Cortex.DTR+#2


title: Rbp4,F,Saline,1week,rep4,b2,b2

description: Library name: AG-2218

value: female

value: Saline


title: C2

description:
sh1_Empty2
UTN_SIN_C2_BIAGIOLI_S12

value: pAIB-empty

value: biol rep 2


title: Hippocampus.DTR-#3

source_name: Hippocampus

value: Hippocampus


title: C4

description:
sh1_Empty4
UTN_SIN_C4_BIAGIOLI_S18

value: biol rep 4


title: spermatozoa, infertility, 710L

description:
no duplicate
710L


title: spermatozoa, infertility, 706L

description:
no duplicate
706L


title: Hippocampus.DTR-#2

value: Whole tissue


title: spermatozoa, infertility, 703L

description:
no duplicate
703L


title: Hippocampus.DTR-#1


title: Cortex.DTR+#3


title: C3

description:
sh1_Empty3
UTN_SIN_C3_BIAGIOLI_S19

value: biol rep 3


title: D4

description:
sh1_sin001_4
UTN_SIN_D4_BIAGIOLI_S17


title: PV,F,Saline,1week,rep1,b2

description: Library name: AG-2242


title: B2

description:
sh1_Unt2
UTN_SIN_B2_BIAGIOLI_S4

value: untransduced


title: spermatozoa, infertility, 709L

description:
no duplicate
709L


title: spermatozoa, infertility, 722L

description:
no duplicate
722L


title: Cortex.DTR+#1

value: Cortex

value: Diphtheria toxin


title: C1

description:
sh1_Empty1
UTN_SIN_C1_BIAGIOLI_S5

value: hiNPCs

value: biol rep 1


title: Rbp4,F,DOI,1week,rep5,b2,b2

description: Library name: AG-2217

value: mouse prefrontal cortex

value: DOI


title: spermatozoa, infertility, 707L

description:
no duplicate
707L

value: spermatozoa

value: infertility


title: B5

description:
sh1_Unt4
UTN_SIN_B4_BIAGIOLI_S16


title: Cux2ERT,F,DOI,1week,rep1,b2

description: Library name: AG-2210

value: female


title: Rbp4,F,DOI,1week,rep4,b2,b2

description: Library name: AG-2216


title: Hippocampus.DTR+#1


title: Cux2ERT,M,Saline,1week,rep2,b2

description: Library name: AG-2214

value: male

value: Saline


title: B3

description:
sh1_Unt3
UTN_SIN_B3_BIAGIOLI_S1


title: Cux2ERT,M,DOI,1week,rep2,b2

description: Library name: AG-2212


title: spermatozoa, infertility, 702L

description:
no duplicate
702L

value: ejaculate


title: Hippocampus.DTR+#3

value: Hippocampus


title: spermatozoa, infertility, 700L

description:
no duplicate
700L


title: Men.DTR+#3

value: Meninges


title: A3

description:
shGFP_Unt3
UTN_SIN_A3_BIAGIOLI_S14

value: Sh-GFP


title: Cux2ERT,F,Saline,1week,rep1,b2

description: Library name: AG-2211


title: Cux2ERT,M,Saline,1month,rep1,b2

description: Library name: AG-2096


title: spermatozoa, infertility, 684L

description:
no duplicate
684L


title: Hippocampus.DTR+#2

value: Whole tissue


title: Men.DTR+#2


title: spermatozoa, proven-fertile, 7

description:
no duplicate
7

value: proven-fertile


title: spermatozoa, infertility, 698L

description:
no duplicate
698L


title: B1

description:
sh1_Unt1
UTN_SIN_B1_BIAGIOLI_S7

value: untransduced


title: A4

description:
shGFP_Unt4
UTN_SIN_A4_BIAGIOLI_S11

value: biol rep 4


title: spermatozoa, infertility, 694L

description:
no duplicate
694L


title: Rbp4,F,Saline,1month,rep5,b2

description: Library name: AG-2075


title: Cux2ERT,M,Saline,1month,rep2,b2

description: Library name: AG-2097


title: Men.DTR+#1

value: Diphtheria toxin


title: A2

description:
shGFP_Unt2
UTN_SIN_A2_BIAGIOLI_S2

value: hiNPCs

value: biol rep 2


title: spermatozoa, infertility, 672L

description:
no duplicate
672L


title: Men.DTR-#1


title: Men.DTR-#2


title: spermatozoa, infertility, 683L

description:
no duplicate
683L


title: Rbp4,M,Saline,1month,rep4,b2

description: Library name: AG-2074

value: mouse prefrontal cortex


title: Snord89_ASO_Rep_2

description: RNA-seq from 293T cells treated with Snord89-targeting ASO, biological replicate 2

data_processing:
Adapter trimming. Command: cutadapt -j 8 --match-read-wildcards --times 3 -e 0.2 --quality-cutoff 6 -m 18 -a AGATCGGAAGAGCACACGTC -a AAAAAAAAAAAAAAAAAAAA -a GGGGGGGGGGGGGGGGGGGG -A AGATCGGAAGAGCGTCGTGT -A TTTTTTTTTTTTTTTTTTTT -O 5
Repeat mapping (takes adapter-trimmed fastq files, and maps to a repeat database to filter repeat elements prior to genome mapping and peak calling, in order to reduce peak artifacts). Command: STAR --runMode alignReads --runThreadN 8 --genomeDir /path/to/homo_sapiens_repbase_v2/ --readFilesCommand zcat --genomeLoad NoSharedMemory --alignEndsType EndToEnd --outSAMunmapped Within --outFilterMultimapNmax 100 --outFilterMultimapScoreRange 1 --outFileNamePrefix /path/to/fi.TrTr.So.Rep --outSAMtype BAM Unsorted --outFilterType BySJout --outBAMcompression 10 --outReadsUnmapped Fastx --outFilterScoreMin 10 --outSAMattrRGline ID:foo --outSAMattributes All --outSAMmode Full --outStd Log --readFilesIn /path/to/fastq.r1.gz /path/to/fastq.r2.gz
Unique genomic mapping (takes repeat-removed raeds and maps to the reference genome). Command: STAR --runMode alignReads --runThreadN 8 --genomeDir /path/to/hg19ref/ --genomeLoad NoSharedMemory --readFilesIn /path/to/fi.TrTr.So.RepUnmapped.out.mate1.s.fq /path/to/fi.TrTr.So.RepUnmapped.out.mate2.s.fq --outSAMunmapped Within --outFilterMultimapNmax 1 --outFilterMultimapScoreRange 1 --outFileNamePrefix /path/to/fi.TrTr.So.rmRep.genomemap --outSAMattributes All --outSAMtype BAM Unsorted --outFilterType BySJout --outReadsUnmapped Fastx --outFilterScoreMin 10 --outSAMattrRGline ID:foo --outStd Log --alignEndsType EndToEnd --outBAMcompression 10 --outSAMmode Full
Alternative splicing was quantitated with rMATS-Turbo (v4.3.0). Command: rmats.py --b1 /path/to/bamfilist.NoASO.txt --b2 /path/to/bamfilist.SNORD89ASO.txt --gtf gencode.v19.annotation.gtf -t paired --libType fr-unstranded --readLength 150 --nthread 8 --od /path/to/rmats_SNORD89_output/ --tmp /path/to/rmats_SNORD89_output/tmp/
Overlapping exons with identical start/stop positions were removed with custom scripts, and overlapping events were removed with the 'subset_jxc' command from the rbp-maps package (https://github.com/YeoLab/rbp-maps)
Assembly: UCSC version hg19
Supplementary files format and content: bigwig files and txt file

growth_protocol: 293T cell lines were cultured in proper medium supplemented with 10% FBS (Cytivia) and antibiotics and maintained in a humidified 5% CO2 incubator at 37°C.

extract_protocol:
RNA was isolated 48 hours after transfection using TRIzol (Thermo).
PolyA-selected RNA-seq libraries were prepared by Novogene and sequenced on the Illumina NovaSeq (150 bp PE) platform. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification.
polyA mRNA-seq

treatment_protocol: 293T cells were transfected with 70 nM of 5-10-5 MOE gapmer ASO (IDT) using Lipofectamine RNAiMAX Transfection Reagent (Thermo). Cell culture media was replaced 24 hours after transfection.

value: Snord89 ASO


title: Rbp4,M,DOI,1month,rep5,b2

description: Library name: AG-2071

value: DOI


title: Rbp4,M,DOI,1month,rep4,b2

description: Library name: AG-2070


title: No_ASO_Rep_2

description: RNA-seq from 293T cells, biological replicate 2


title: spermatozoa, infertility, 661L

description:
no duplicate
661L

value: spermatozoa

value: infertility


title: spermatozoa, infertility, 666L

description:
no duplicate
666L


title: spermatozoa, infertility, 651L

description:
no duplicate
651L


title: No_ASO_Rep_1

description: RNA-seq from 293T cells, biological replicate 1


title: Men.72.DTR+#1


title: Snord89_ASO_Rep_1

description: RNA-seq from 293T cells treated with Snord89-targeting ASO, biological replicate 1


title: RB1

city: Ipswich

first: Sriharsa

last: Pradhan

address: 240 County Road

institute: New England Biolabs

library_source: genomic

data_processing:
Library Strategy: NEED-seq
Adaptor and low-quality sequences were trimmed from paired-end sequencing reads using Trim Galore v 0.5.0
NEED-seq:Trimmed read pairs were mapped to the reference genome (human: hg38) using Bowtie2 v. 2.3.4.3. PCR duplicates and mitochondrial reads were removed using Picard tools and samtools. The aligned bam files of technical replicates were merged using samtools. Open chromatin regions were identified by calling narrow peaks and Closed chromatin were identified by calling broad peaks using MACS2.
EM-seq: Trimmed read pairs were mapped to the reference genome (human: hg38) using Bwa-meth. PCR duplicates and mitochondrial reads were removed using samblaster and samtools.
ChIP-seq: Trimmed read pairs were mapped to the reference genome (human: hg38) using Bowtie2 v. 2.3.4.3. PCR duplicates and mitochondrial reads were removed using Picard tools and samtools. The aligned bam files of technical replicates were merged using samtools.  Open chromatin regions were identified by calling narrow peaks and Closed chromatin were identified by calling broad peaks using MACS2 where Input were used as control
Hi-C: HiC data were analysed using HicPro.
Assembly: hg38
Supplementary files format and content: Bigwigs were generated using bins using deeptools bamCoverage with the following parameters: --normalizeUsing RPKM

molecule: genomic DNA

growth_protocol: HT1080 cells were grown in 6 well plates until 70-80% confluency. After one wash with 1XPBS, cells were crosslinked in 1XPBS with 4% formaldehyde (Thermo Fisher Scientific # 28908) for 10 min at RT. K562 crosslinked cells with 4% formaldehyde were decrosslinked overnight at 58°C in 1X PBS. Cytoplasmic extraction was performed for 30 min with end-over-end mixing at 4°C. After 300 g centrifugation at 4°C for 5 min, cell pellet was washed once with 1X PBS and nuclei concentration was defined using Luna automated cell counter.

extract_protocol:
Cells were fixed with 1% and 4% formaldehyde
HT1080 cells were grown in 6 well plates until 70-80% confluency. After one wash with 1X PBS (GIBCO # 770011-044), cells were crosslinked in 1X PBS with 4% formaldehyde (Thermo Fisher Scientific # 28908) for 10 min at room temperature (RT). In some crosslinking conditions, 1% formaldehyde was used. Crosslink was quenched using 0.25 M Glycine for 5 min at RT. Cells were de-crosslinked overnight at 58°C in 1X PBS. Cytoplasm was extracted for 10-30 min on ice using cytosolic buffer (15mM Tris-HCl, pH7.5, 5mM MgCl2, 60mM KCl, 0.5mM DTT, 15mM NaCl, 300mM sucrose, and 1% NP40) with occasional agitation. After 1X PBS wash, cells were incubated with 1X PBS / 5% BSA / 0.3% Triton™ X-100 for 1 hr at RT with gentle rocking. Primary antibodies were added (1/400 dilution per well) and incubated overnight at 4°C with gentle rocking. After 3 washes with 1X T-PBS (PBS + 0.1% Tween 20) for 5 min at RT, secondary antibodies were added , Alexa Fluor® 488 conjugated goat anti-Mouse IgG (H+L) Superclonal™ or goat anti-Rabbit IgG (H+L) Superclonal™ (corresponding to species of primary antibody; Thermo Fisher # A-28175 and A-27034 respectively) at a dilution of 1:1000 in PBS / 0.3% Triton™ X-100/ 5% BSA for 1 hr at RT with gentle rocking. After 3 washes with 1X T-PBS for 5 min at RT, cells were incubated for 1 hr at RT with 1 µl of Nt.CviPII-pGL (NEB, 0.5 units) in 800 µl binding buffer per well (20 mM potassium phosphate pH 7.0, 20 mM NaCl, 0.3% Triton™ X-100). In some experiments, heparin (Sigma-Aldrich # H3149) was added at different concentrations to prevent non-specific DNA binding. Plates were then washed 3 times with washing buffer (20 mM potassium phosphate buffer  pH 7.0, 500 mM NaCl, 0.3% Triton™ X-100) for 15 min at RT. Cells were incubated with 10 units of DNA Polymerase I (NEB # M0209S), 5 mM of dNTP mix including Biotin-14-dCTP (Thermo Fisher # 19518018), 5-methyl-dCTP (NEB # N0356S), dATP, dTTP and dGTP for 1 hr at 37°C in 800 µl of 1X NEBuffer 2 per well to allow nick translation to occur. Plates were then washed 3 times with washing buffer (20 mM potassium phosphate buffer, pH 7.0, 500 mM NaCl, 0.3% Triton™ X-100) for 15 min at RT with an additional wash with 1X PBS. Cells were then covered with 100 µl of 1X PBS and DNA was extracted using Monarch genomic DNA purification kit (NEB # T3010S) according to manufacturer’s recommendations. After DNA purification, 100-500 ng of genomic DNA was fragmented using 2 units of Nt.CviPII (NEB # R0626S) in 500  µl of 1X NEBuffer 2 at 37°C overnight. Nt.CviPII was inactivated at 65°C for 20 min. NaCl 5M stock was added to reach 1 M final concentration along with 30 µl of Streptavidin magnetic beads (NEB # S1420S) for 2 hrs with end-over-end mixing at 4°C to capture biotinylated DNA fragments. Streptavidin bound DNA beads were washed twice with 1X TE buffer supplemented with 2 M NaCl and 0.05 % Triton™ X-100. A third wash was performed with 1X TE buffer. Beads were resuspended in 50 µl of TE buffer and libraries were made on beads using NEBNext®Ultra™ II DNA Library Prep Kit for Illumina® (NEB # E7645S) according to manufacturer’s protocol. 8 to 12 PCR cycles were used to amplify barcoded DNA. NEED-seq protocol described above was used with some modifications. Briefly, K562 crosslinked cells with 4% formaldehyde were decrosslinked overnight at 58°C in 1X PBS. Cytoplasmic extraction was performed for 30 min with end-over-end mixing at 4°C. After 300 x g centrifugation at 4°C for 5 min, cell pellet was washed once with 1X PBS and nuclei number was calculated using Luna automated cell counter. Nuclei resuspended in 1X PBS were then captured by adding 10 µl of preactivated Concanavalin A magnetic beads (Cell Signaling Technology # 93569) for 10 min with end-over-end mixing at RT. Bead-bound nuclei were incubated with 1X PBS including 0.3 % Triton X-100 and 5% BSA for 1 hr at RT. Primary antibodies were added (1/400 final dilution) and incubated overnight with end-over-end mixing at 4°C. After 3 washes with 1X T-PBS for 5 min at RT, blocking buffer was added with anti-mouse IgG or anti-rabbit IgG coupled with Alexa 488 for 1h at RT with gentle rocking. After 3 washes with 1X T-PBS for 5 min at RT, beads were resuspended in 1 ml binding buffer (as described above) including 1 l of fusion enzyme Nt.CviPII-pGL (NEB) for 1 hr at RT. Beads were then washed 3 times with washing buffer (as described above) for 15 min at RT. After 1 wash with 1X PBS, beads were incubated with 10 units of DNA Polymerase I, 5 M of dNTP mix including Biotin-14-dCTP, 5-methyl-dCTP, dATP, dTTP and dGTP for 1 hr at 37°C in 800 µl of 1X NEBuffer 2 with end-over-end mixing. After 3 washes with washing buffer for 15 min at RT, beads were resuspended in 50 mM Tris-Cl, pH 7.5, 1 % SDS, 200 mM NaCl and 2 µl of proteinase K (NEB # P8107S) and incubated overnight at 65°C to lyse nuclei bound to the beads and DNA was extracted using phenol-chloroform and precipitated using glycogen. 200 ng of DNA was fragmented using Nt.CviPII and used to make libraries as described above. For low number of cells, barcoded DNA was amplified using ~18 PCR cycles. To determine the dynamic range of NEED-seq, cells were serially diluted after the labeling reaction. In this modified NEED-seq method, HT1080 cells were grown on 6 well plate to 80% confluency. Cells were incubated for 10 min at 4°C using CSK buffer (10 mM PIPES pH 6.8, 300 mM sucrose, 100 mM NaCl, 1.5 mM MgCl2 and 0.5 % Triton X-100) followed by crosslinking with 4% formaldehyde for 10 min at RT. Glycine was used to quench formaldehyde for 5 min at RT (final concentration 0.25M). After 1X PBS wash, blocking buffer was added to cells for 1h at RT (5% BSA, 1X PBS and 0.3% Triton X-100). Different antibodies (see Table S2) were then added at dilutions recommended by the manufacturer and incubated with cells for 16 hrs at 4°C. After 3 washes with T-PBS (PBS with 0.1 % Tween 20) for 5 min at RT, blocking buffer was added with anti-mouse IgG or anti-rabbit IgG coupled with Alexa 488 for 1h at RT. After 3 washes with T-PBS for 5 min at RT, NEED-seq binding buffer (20 mM KPO4, 150 mM NaCl and 0.3% TritonX-100) was added along with Nt.CviPII-pGL enzyme (1 unit per well) for 1h at 37°C. After 3 washes with NEED-seq washing buffer (20 mM KPO4, 500 mM NaCl and 1% TritonX-100) for 15 min at RT, 1X NEBuffer2.1 was added for 1h at 37°C to allow Nt.CviPII-pGL activity to fragment the targeted DNA. After 1 wash with 1X PBS, genomic DNA extraction was performed using Monarch genomic DNA purification kit (6 wells pooled per antibody) according to NEB recommendations with a minor modification which included a lysis step at 56°C for 1h instead of 10 min to reverse DNA crosslinking. 0.1 to 1 µg of genomic DNA was used for size selection using first 0.4x volume of NEBNext Sample Purification beads with 10 min incubation at RT. Supernatant containing 200-500 bp DNA sizes was separated from beads using 12-tube magnetic separation rack (NEB # S1509S) and collected. An additional 0.4x volume of NEBNext Sample Purification beads was added to the supernatant and incubated for 10 min at RT. After 2 washes with 80% EtOH, DNA was eluted form beads with 50 µl of low TE buffer and DNA libraries were prepared using NEBNext Ultra II DNA Library Prep (NEB # E7103L) with some modifications. Briefly, end repair/dA tailing was performed on size selected DNA and NEBNext Illumina adaptor (1/10 dilution) were ligated for 2 hrs at RT. USER enzyme (NEB # E7103L) was incubated for 20 min at 37°C and another round of library size selection was performed as described above. The library was amplified using 13 PCR cycles. PCR products were purified using 0.9x volume of NEBNext Sample Purification beads. 1 nM of size selected DNA library was sequenced on Novaseq 6000. HT1080 cells (0.5x10e6) were crosslinked with 4% formaldehyde for 10 min at RT and quenched with 1.5 M Tris-HCl (pH 8) for 5 min at RT. After 1X PBS wash, cells were permeabilized with Hi-C lysis buffer (10 mM Tris-HCl (pH 8), 10 mM NaCl and 0.2% NP40) for 30 min at 4°C, washed with 1X NEBuffer2 and incubated for 10 min at 58°C with 1X NEBuffer2, 0.3% SDS and 1 mM DTT. To quench SDS, Triton X-100 was added (1% final concentration per well) for 15 min at 37°C. 400 U of MboI (NEB # R0147M) were added and incubated for 16 hrs at 37°C. Enzyme was inactivated at 58°C for 20 min. Supernatant was discarded and DNA polymerase I mix containing  NEBuffer2.1 1X (NEB # B7002S), 30 µM of dNTPs (including dCTP/dGTP/dTTP (NEB # N0441S/ N0442S/N0443S respectively) with biotin-14-dATP (ThermoFisher Scientific # 19524016)) and 50 U of DNA polymerase I (NEB # M0210M) was added and incubated for 4 hrs at RT. Ligase mix was added and incubated for 16 hrs at RT. Ligase mix contains 2000 U of T4 DNA ligase (M0202M NEB), 140 µg of recombinant albumin (NEB # B9200S),  1X T4 DNA ligase buffer (NEB # B0202S) and 1 % Triton X-100. After ligation, supernatant was discarded, and cells were washed using 1X PBS. Genomic DNA was extracted from the wells using Monarch Genomic DNA purification Kit (NEB # T3010L). To remove biotin from un-ligated DNA ends, 10 µg of genomic DNA was incubated with 30 U of T4 DNA Polymerase (NEB # M0203L), 25 µM dATP/dGTP (NEB # N0440S/N0442S respectively) in 1X NEBuffer2 for 4 hrs at 20°C. Enzyme was inactivated for 20 min at 75°C. DNA was purified using 1X volume of NEBNext Sample Purification beads (NEB # E6178S) and sheared to 300 bp using Covaris (program : repeat/iterations # 7; repeat process treatment duration 10s; peak power 50W; duty factor 20%; cycles per burst 1000; total treatment time per sample 70s). To capture biotinylated DNA, DNA was incubated with NEB Magnetic Streptavidin beads (NEB # S1420S) for 30 min at RT in 10 mM Tris-HCl (pH 7.5) supplemented with 1 M NaCl. Beads were washed once with 10 mM Tris-HCl (pH 7.5) supplemented with 1 M NaCl and once with low TE buffer. Beads were resuspended in 50 µl of low TE buffer and DNA libraries were prepared on beads using NEBNext Ultra II DNA Library Prep (NEB # E7103L) with some modifications. Briefly, end repair/dA tailing was performed on beads and NEBNext Illumina adaptor (1/10 dilution) were ligated for 2 hrs at RT. 3 µl of USER (NEB # E7103L) was added for 20 min at 37°C. Beads were then washed once with 10 mM Tris-HCl (pH 7.5) supplemented with 1 M of NaCl and once with low TE. Beads were resuspended in 15 µl of low TE. 14 PCR cycles were performed. Supernatant containing PCR products were separated from beads using 12-tube magnetic separation rack (NEB # S1509S) and purified using 0.9x volume of NEBNext Sample Purification beads. 1 nM of DNA Hi-C library was loaded on Novaseq 6000.

value: fibrosarcoma cells

tag: condition


title: Men.72.DTR-#2


title: PV,F,DOI,1month,rep2,b2

description: Library name: AG-2031

value: PV

value: female


title: spermatozoa, proven-fertile, 6

description:
no duplicate
6

value: ejaculate


title: PV,M,Saline,1month,rep1,b2

description: Library name: AG-2029

value: male

value: Saline


title: spermatozoa, proven-fertile, 27

description:
no duplicate
27

value: proven-fertile


title: p53


title: Men.72.DTR-#3

value: Meninges

value: Whole tissue


title: PV,M,DOI,1month,rep1,b2

description: Library name: AG-2028


title: Rbp4,F,Saline,1week,rep2,b1

description: Library name: AG-2396


title: Men.72.DTR-#1

value: Diphtheria toxin


title: Men.12.DTR+#2


title: spermatozoa, proven-fertile, 38

description:
no duplicate
38


title: NPAT

value: NPAT


title: A1

description:
shGFP_Unt1
UTN_SIN_A1_BIAGIOLI_S15

value: hiNPCs

value: Sh-GFP

value: untransduced

value: biol rep 1


title: c-JUN

value: c-JUN


title: spermatozoa, proven-fertile, 23

description:
no duplicate
23

value: spermatozoa


title: c-FOS

value: fibrosarcoma cells

value: c-FOS


title: PV,F,Saline,1month,rep2,b2

description: Library name: AG-2034

value: mouse prefrontal cortex

value: PV

value: female


title: spermatozoa, proven-fertile, 26

description:
no duplicate
26

value: ejaculate


title: IgG+2

value: IgG


title: Men.72.DTR+#2

value: Meninges


title: MDA-B-231 cells, EGFP negative 2

city: CAMBRIDGE

first: Satomi

last: Hirose

address: 500 Technology Square

department: Mechanical Engineering

institute: Massachusetts Institute of Technology

description: instrument: Singular G4

data_processing:
The raw reads were demultiplexed based on the tag and mapped to build GRCh37/hg19 of the human reference genome.
Next, the mapped reads were quantified using the featureCount in Subread.
Assembly: GRCh37/hg19
Supplementary files format and content: text files include raw counts for each sample

source_name: Breast

growth_protocol: MDA-MB-231 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U mL−1 penicillin, and 100 µg mL−1 streptomycin in a humidified atmosphere with 5% CO2 at 37C

extract_protocol:
MDA-MB-231 cells were lysed using RNeasy Plus Mini Kit and kept at −80°C until needed.
PolyA selection and stranded RNA-seq library preparation were performed with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina

value: Breast

value: Triple negative breast cancer cells

value: WT

tag: transfection

value: H2B-EGFP, CMV-RFP

tag: group

value: EGFP negative

tag: status

value: increased extravasation


title: Men.12.DTR-#2

value: Whole tissue

value: Diphtheria toxin


title: Rbp4,F,DOI,1week,rep3,b1

description: Library name: AG-2394

value: DOI


title: Rbp4,F,Saline,1week,rep1,b1

description: Library name: AG-2395

value: Saline


title: Rbp4,F,DOI,1week,rep2,b1

description: Library name: AG-2391


title: spermatozoa, proven-fertile, 21

description:
no duplicate
21

value: spermatozoa

value: proven-fertile


title: Men.12.DTR-#1


title: HP.72.DTR+#1

value: Hippocampus


title: Men.12.DTR+#1


title: Rbp4,F,Saline,1week,rep3,b1

description: Library name: AG-2397

value: mouse prefrontal cortex

value: female


title: spermatozoa, proven-fertile, 13

description:
no duplicate
13

value: ejaculate


title: HP.72.DTR+#2


title: Rbp4,M,DOI,1week,rep1,b1

description: Library name: AG-2388

value: male


title: Rbp4,F,Saline,1month,rep3,b1

description: Library name: AG-2077


title: MDA-B-231 cells, EGFP negative 1

description: instrument: Singular G4

value: Breast


title: spermatozoa, proven-fertile, 18

description:
no duplicate
18


title: MDA-B-231 cells, EGFP positive 1

description: instrument: Singular G4

value: Triple negative breast cancer cells

value: EGFP positive

value: no increased extravasation


title: spermatozoa, proven-fertile, 12

description:
no duplicate
12


title: spermatozoa, proven-fertile, 11

description:
no duplicate
11


title: spermatozoa, proven-fertile, 22

description:
no duplicate
22


title: MDA-B-231 cells, EGFP positive 2

description: instrument: Singular G4

value: WT

value: H2B-EGFP, CMV-RFP


title: Rbp4,F,Saline,1month,rep1,b1

description: Library name: AG-2068


title: HP.12.DTR+#1

value: Whole tissue


title: HP.72.DTR-#1

value: Diphtheria toxin


title: Rbp4,M,Saline,1month,rep2,b1

description: Library name: AG-2073


title: HP.72.DTR-#2


title: H3K4me3_MNase+4C (Size Selection)

data_processing:
Adaptor and low-quality sequences were trimmed from paired-end sequencing reads using Trim Galore v 0.5.0
NEED-seq:Trimmed read pairs were mapped to the reference genome (human: hg38) using Bowtie2 v. 2.3.4.3. PCR duplicates and mitochondrial reads were removed using Picard tools and samtools. The aligned bam files of technical replicates were merged using samtools. Open chromatin regions were identified by calling narrow peaks and Closed chromatin were identified by calling broad peaks using MACS2.
EM-seq: Trimmed read pairs were mapped to the reference genome (human: hg38) using Bwa-meth. PCR duplicates and mitochondrial reads were removed using samblaster and samtools.
ChIP-seq: Trimmed read pairs were mapped to the reference genome (human: hg38) using Bowtie2 v. 2.3.4.3. PCR duplicates and mitochondrial reads were removed using Picard tools and samtools. The aligned bam files of technical replicates were merged using samtools.  Open chromatin regions were identified by calling narrow peaks and Closed chromatin were identified by calling broad peaks using MACS2 where Input were used as control
Hi-C: HiC data were analysed using HicPro.
Assembly: hg38
Supplementary files format and content: Bigwigs were generated using bins using deeptools bamCoverage with the following parameters: --normalizeUsing RPKM
Library strategy: NEED-seq

growth_protocol: HT1080 and HCT116 cells were grown in 6 well plates until 70-80% confluency. After one wash with 1XPBS, cells were crosslinked in 1XPBS with 4% formaldehyde (Thermo Fisher Scientific # 28908) for 10 min at RT. K562 crosslinked cells with 4% formaldehyde were decrosslinked overnight at 58°C in 1X PBS. Cytoplasmic extraction was performed for 30 min with end-over-end mixing at 4°C. After 300 g centrifugation at 4°C for 5 min, cell pellet was washed once with 1X PBS and nuclei concentration was defined using Luna automated cell counter.

extract_protocol:
Cells were fixed with 1% and 4% formaldehyde
HT1080 and HCT116 cells were grown in 6 well plates until 70-80% confluency. After one wash with 1X PBS (GIBCO # 770011-044), cells were crosslinked in 1X PBS with 4% formaldehyde (Thermo Fisher Scientific # 28908) for 10 min at room temperature (RT). In some crosslinking conditions, 1% formaldehyde was used. Crosslink was quenched using 0.25 M Glycine for 5 min at RT. Cells were de-crosslinked overnight at 58°C in 1X PBS. Cytoplasm was extracted for 10-30 min on ice using cytosolic buffer (15mM Tris-HCl, pH7.5, 5mM MgCl2, 60mM KCl, 0.5mM DTT, 15mM NaCl, 300mM sucrose, and 1% NP40) with occasional agitation. After 1X PBS wash, cells were incubated with 1X PBS / 5% BSA / 0.3% Triton™ X-100 for 1 hr at RT with gentle rocking. Primary antibodies were added (1/400 dilution per well) and incubated overnight at 4°C with gentle rocking. After 3 washes with 1X T-PBS (PBS + 0.1% Tween 20) for 5 min at RT, secondary antibodies were added , Alexa Fluor® 488 conjugated goat anti-Mouse IgG (H+L) Superclonal™ or goat anti-Rabbit IgG (H+L) Superclonal™ (corresponding to species of primary antibody; Thermo Fisher # A-28175 and A-27034 respectively) at a dilution of 1:1000 in PBS / 0.3% Triton™ X-100/ 5% BSA for 1 hr at RT with gentle rocking. After 3 washes with 1X T-PBS for 5 min at RT, cells were incubated for 1 hr at RT with 1 µl of Nt.CviPII-pGL (NEB, 0.5 units) in 800 µl binding buffer per well (20 mM potassium phosphate pH 7.0, 20 mM NaCl, 0.3% Triton™ X-100). In some experiments, heparin (Sigma-Aldrich # H3149) was added at different concentrations to prevent non-specific DNA binding. Plates were then washed 3 times with washing buffer (20 mM potassium phosphate buffer  pH 7.0, 500 mM NaCl, 0.3% Triton™ X-100) for 15 min at RT. Cells were incubated with 10 units of DNA Polymerase I (NEB # M0209S), 5 mM of dNTP mix including Biotin-14-dCTP (Thermo Fisher # 19518018), 5-methyl-dCTP (NEB # N0356S), dATP, dTTP and dGTP for 1 hr at 37°C in 800 µl of 1X NEBuffer 2 per well to allow nick translation to occur. Plates were then washed 3 times with washing buffer (20 mM potassium phosphate buffer, pH 7.0, 500 mM NaCl, 0.3% Triton™ X-100) for 15 min at RT with an additional wash with 1X PBS. Cells were then covered with 100 µl of 1X PBS and DNA was extracted using Monarch genomic DNA purification kit (NEB # T3010S) according to manufacturer’s recommendations. After DNA purification, 100-500 ng of genomic DNA was fragmented using 2 units of Nt.CviPII (NEB # R0626S) in 500  µl of 1X NEBuffer 2 at 37°C overnight. Nt.CviPII was inactivated at 65°C for 20 min. NaCl 5M stock was added to reach 1 M final concentration along with 30 µl of Streptavidin magnetic beads (NEB # S1420S) for 2 hrs with end-over-end mixing at 4°C to capture biotinylated DNA fragments. Streptavidin bound DNA beads were washed twice with 1X TE buffer supplemented with 2 M NaCl and 0.05 % Triton™ X-100. A third wash was performed with 1X TE buffer. Beads were resuspended in 50 µl of TE buffer and libraries were made on beads using NEBNext®Ultra™ II DNA Library Prep Kit for Illumina® (NEB # E7645S) according to manufacturer’s protocol. 8 to 12 PCR cycles were used to amplify barcoded DNA. NEED-seq protocol described above was used with some modifications. Briefly, K562 crosslinked cells with 4% formaldehyde were decrosslinked overnight at 58°C in 1X PBS. Cytoplasmic extraction was performed for 30 min with end-over-end mixing at 4°C. After 300 x g centrifugation at 4°C for 5 min, cell pellet was washed once with 1X PBS and nuclei number was calculated using Luna automated cell counter. Nuclei resuspended in 1X PBS were then captured by adding 10 µl of preactivated Concanavalin A magnetic beads (Cell Signaling Technology # 93569) for 10 min with end-over-end mixing at RT. Bead-bound nuclei were incubated with 1X PBS including 0.3 % Triton X-100 and 5% BSA for 1 hr at RT. Primary antibodies were added (1/400 final dilution) and incubated overnight with end-over-end mixing at 4°C. After 3 washes with 1X T-PBS for 5 min at RT, blocking buffer was added with anti-mouse IgG or anti-rabbit IgG coupled with Alexa 488 for 1h at RT with gentle rocking. After 3 washes with 1X T-PBS for 5 min at RT, beads were resuspended in 1 ml binding buffer (as described above) including 1 l of fusion enzyme Nt.CviPII-pGL (NEB) for 1 hr at RT. Beads were then washed 3 times with washing buffer (as described above) for 15 min at RT. After 1 wash with 1X PBS, beads were incubated with 10 units of DNA Polymerase I, 5 M of dNTP mix including Biotin-14-dCTP, 5-methyl-dCTP, dATP, dTTP and dGTP for 1 hr at 37°C in 800 µl of 1X NEBuffer 2 with end-over-end mixing. After 3 washes with washing buffer for 15 min at RT, beads were resuspended in 50 mM Tris-Cl, pH 7.5, 1 % SDS, 200 mM NaCl and 2 µl of proteinase K (NEB # P8107S) and incubated overnight at 65°C to lyse nuclei bound to the beads and DNA was extracted using phenol-chloroform and precipitated using glycogen. 200 ng of DNA was fragmented using Nt.CviPII and used to make libraries as described above. For low number of cells, barcoded DNA was amplified using ~18 PCR cycles. To determine the dynamic range of NEED-seq, cells were serially diluted after the labeling reaction. In this modified NEED-seq method, HT1080 cells were grown on 6 well plate to 80% confluency. Cells were incubated for 10 min at 4°C using CSK buffer (10 mM PIPES pH 6.8, 300 mM sucrose, 100 mM NaCl, 1.5 mM MgCl2 and 0.5 % Triton X-100) followed by crosslinking with 4% formaldehyde for 10 min at RT. Glycine was used to quench formaldehyde for 5 min at RT (final concentration 0.25M). After 1X PBS wash, blocking buffer was added to cells for 1h at RT (5% BSA, 1X PBS and 0.3% Triton X-100). Different antibodies (see Table S2) were then added at dilutions recommended by the manufacturer and incubated with cells for 16 hrs at 4°C. After 3 washes with T-PBS (PBS with 0.1 % Tween 20) for 5 min at RT, blocking buffer was added with anti-mouse IgG or anti-rabbit IgG coupled with Alexa 488 for 1h at RT. After 3 washes with T-PBS for 5 min at RT, NEED-seq binding buffer (20 mM KPO4, 150 mM NaCl and 0.3% TritonX-100) was added along with Nt.CviPII-pGL enzyme (1 unit per well) for 1h at 37°C. After 3 washes with NEED-seq washing buffer (20 mM KPO4, 500 mM NaCl and 1% TritonX-100) for 15 min at RT, 1X NEBuffer2.1 was added for 1h at 37°C to allow Nt.CviPII-pGL activity to fragment the targeted DNA. After 1 wash with 1X PBS, genomic DNA extraction was performed using Monarch genomic DNA purification kit (6 wells pooled per antibody) according to NEB recommendations with a minor modification which included a lysis step at 56°C for 1h instead of 10 min to reverse DNA crosslinking. 0.1 to 1 µg of genomic DNA was used for size selection using first 0.4x volume of NEBNext Sample Purification beads with 10 min incubation at RT. Supernatant containing 200-500 bp DNA sizes was separated from beads using 12-tube magnetic separation rack (NEB # S1509S) and collected. An additional 0.4x volume of NEBNext Sample Purification beads was added to the supernatant and incubated for 10 min at RT. After 2 washes with 80% EtOH, DNA was eluted form beads with 50 µl of low TE buffer and DNA libraries were prepared using NEBNext Ultra II DNA Library Prep (NEB # E7103L) with some modifications. Briefly, end repair/dA tailing was performed on size selected DNA and NEBNext Illumina adaptor (1/10 dilution) were ligated for 2 hrs at RT. USER enzyme (NEB # E7103L) was incubated for 20 min at 37°C and another round of library size selection was performed as described above. The library was amplified using 13 PCR cycles. PCR products were purified using 0.9x volume of NEBNext Sample Purification beads. 1 nM of size selected DNA library was sequenced on Novaseq 6000. HT1080 cells (0.5x10e6) were crosslinked with 4% formaldehyde for 10 min at RT and quenched with 1.5 M Tris-HCl (pH 8) for 5 min at RT. After 1X PBS wash, cells were permeabilized with Hi-C lysis buffer (10 mM Tris-HCl (pH 8), 10 mM NaCl and 0.2% NP40) for 30 min at 4°C, washed with 1X NEBuffer2 and incubated for 10 min at 58°C with 1X NEBuffer2, 0.3% SDS and 1 mM DTT. To quench SDS, Triton X-100 was added (1% final concentration per well) for 15 min at 37°C. 400 U of MboI (NEB # R0147M) were added and incubated for 16 hrs at 37°C. Enzyme was inactivated at 58°C for 20 min. Supernatant was discarded and DNA polymerase I mix containing  NEBuffer2.1 1X (NEB # B7002S), 30 µM of dNTPs (including dCTP/dGTP/dTTP (NEB # N0441S/ N0442S/N0443S respectively) with biotin-14-dATP (ThermoFisher Scientific # 19524016)) and 50 U of DNA polymerase I (NEB # M0210M) was added and incubated for 4 hrs at RT. Ligase mix was added and incubated for 16 hrs at RT. Ligase mix contains 2000 U of T4 DNA ligase (M0202M NEB), 140 µg of recombinant albumin (NEB # B9200S),  1X T4 DNA ligase buffer (NEB # B0202S) and 1 % Triton X-100. After ligation, supernatant was discarded, and cells were washed using 1X PBS. Genomic DNA was extracted from the wells using Monarch Genomic DNA purification Kit (NEB # T3010L). To remove biotin from un-ligated DNA ends, 10 µg of genomic DNA was incubated with 30 U of T4 DNA Polymerase (NEB # M0203L), 25 µM dATP/dGTP (NEB # N0440S/N0442S respectively) in 1X NEBuffer2 for 4 hrs at 20°C. Enzyme was inactivated for 20 min at 75°C. DNA was purified using 1X volume of NEBNext Sample Purification beads (NEB # E6178S) and sheared to 300 bp using Covaris (program : repeat/iterations # 7; repeat process treatment duration 10s; peak power 50W; duty factor 20%; cycles per burst 1000; total treatment time per sample 70s). To capture biotinylated DNA, DNA was incubated with NEB Magnetic Streptavidin beads (NEB # S1420S) for 30 min at RT in 10 mM Tris-HCl (pH 7.5) supplemented with 1 M NaCl. Beads were washed once with 10 mM Tris-HCl (pH 7.5) supplemented with 1 M NaCl and once with low TE buffer. Beads were resuspended in 50 µl of low TE buffer and DNA libraries were prepared on beads using NEBNext Ultra II DNA Library Prep (NEB # E7103L) with some modifications. Briefly, end repair/dA tailing was performed on beads and NEBNext Illumina adaptor (1/10 dilution) were ligated for 2 hrs at RT. 3 µl of USER (NEB # E7103L) was added for 20 min at 37°C. Beads were then washed once with 10 mM Tris-HCl (pH 7.5) supplemented with 1 M of NaCl and once with low TE. Beads were resuspended in 15 µl of low TE. 14 PCR cycles were performed. Supernatant containing PCR products were separated from beads using 12-tube magnetic separation rack (NEB # S1509S) and purified using 0.9x volume of NEBNext Sample Purification beads. 1 nM of DNA Hi-C library was loaded on Novaseq 6000.

value: fibrosarcoma cells


title: HP.12.DTR-#2


title: HP.12.DTR-#1


title: Rbp4,F,DOI,1month,rep3,b1

description: Library name: AG-2069

value: DOI


title: HP.12.DTR+#2


title: H3K4me3_MNase+37C (Size Selection)


title: Meninges.aCD25#1

value: Meninges

value: anti-CD25 mAb


title: H3K27me3_MNase+4C (Size Selection)


title: HepG2_si-Non Target

type: RNA

city: Yoshida-gun

first: Takuto

last: Nosaka

state: Fukui

address: 23-3 Matsuoka, Eiheiji-cho

department: Second Department of Internal Medicine

country: Japan

institute: University of Fukui

hyb_protocol: 0.6 μg of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25 μl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer’s instructions. On completion of the fragmentation reaction, 25 μl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to SurePrint G3 Human GE 8x60K Microarray Ver3.0 (Agilent Technologies) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent Technologies) and 1 minute with 37°C GE Wash buffer 2 (Agilent Technologies).

scan_protocol: Slides were scanned immediately after washing on the Agilent SureScan Microarray Scanner (G2600D) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green PMT is set to 100%).

data_processing: The scanned images were analyzed with Feature Extraction Software 12.0.3.1 (Agilent Technologies) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.

source_name: HepG2.D11 cell

label_protocol: Cyanine3 (Cy3) labeled cRNA was prepared from 0.1 μg Total RNA using the Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-2000 Spectrophotometer.

growth_protocol: A monolayer of primary human hepatocytes derived from chimeric mice with human liver was obtained from PhoenixBio Co., Lid (Hiroshima, Japan) and grown in BioCoatTM Collagen I white plate (Corning Life Science, Tewksburry, MA, USA). Hepatocytes were cultured in dHCGM medium consisting of Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St Lois, MO, USA) with 10% FBS, 20 mM HEPES (Life Technologies/Gibco), 44 mM NaHCO3 (WAKO, Osaka, Japan), 100 U/ml penicillin G, 100 μg/ml streptomycin, 15 μg/ml L-proline (WAKO), 0.25 μg/ml Insulin (Sigma-Aldrich), 50 nM dexamethasone (Sigma-Aldrich), 5 ng/ml epidermal growth factor (Sigma-Aldrich), 0.1 mM L-ascorbic acid 2-phosphate (WAKO), and 2% dimethyl sulfoxide (Sigma-Aldrich). The human liver cancer cell line HepG2 was obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured with RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine, 1 μmol/L sodium pyruvate, 0.1 mmol/L nonessential amino acids, and 100 U/mL penicillin/50 μg/mL streptomycin (Gibco, Grand Island, NY, USA) at 37°C with 5% CO2.

extract_protocol: RNAs were extracted with TRIZol reagent (Thermo Fisher Scientific) and cDNA was synthesized with High Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City CA, USA), following the manufacturer’s instructions.

treatment_protocol: HBV for in vitro viral infection was obtained from the culture medium of HepG2 transfected with HBV plasmids. HBV DNA was isolated using SMITEST EX-R&D (MBL; Medical & Biological laboratories Co., LTD, Nagano, Japan) according to the manufacture’s protocol, and the amount of HBV DNA was determined by real-time PCR. PXB cells were infected with the collected HBV at same equivalents in the presence of 4% polyethylene glycol 8000 (PEG8000, Sigma-Aldrich). siRNA was transfected into the PXB cells on day 0 and HBV was added on day 4 and PXB cells were collected on day 13. The 1.3-mer HBV genomes [genotype C2, both basal core promoter (BCP) A1762T/G1764A mutation and precore G1896A mutation, accession number AB819615] were inserted into the KpnΙ and NotΙ restriction sites of pBluescript II SK (Agilent Technologies, Santa Clara, USA). Plasmid was transfected into HepG2 cells using LipofectamineTM LTX Reagent (Thermo Fisher Scientific, Waltham, MA, USA). HBV transfected HepG2 cells that were confirmed to stably produce HBsAg in the supernatant were called HepG2.D11 clones. HepG2.D11 cells were seeded on day 0 and siRNA was transfected at medium change on day 1. HepG2.D11 cells were collected on day 5.

value: HBV transfected, si-Non target

tag: tag

value: HepG2 cell


title: Rbp4,M,DOI,1month,rep1,b1

description: Library name: AG-2064

value: mouse prefrontal cortex


title: LaminB2

extract_protocol:
Cells were fixed with 1% and 4% formaldehyde
HT1080 and HCT116 cells were grown in 6 well plates until 70-80% confluency. After one wash with 1X PBS (GIBCO # 770011-044), cells were crosslinked in 1X PBS with 4% formaldehyde (Thermo Fisher Scientific # 28908) for 10 min at room temperature (RT). In some crosslinking conditions, 1% formaldehyde was used. Crosslink was quenched using 0.25 M Glycine for 5 min at RT. Cells were de-crosslinked overnight at 58°C in 1X PBS. Cytoplasm was extracted for 10-30 min on ice using cytosolic buffer (15mM Tris-HCl, pH7.5, 5mM MgCl2, 60mM KCl, 0.5mM DTT, 15mM NaCl, 300mM sucrose, and 1% NP40) with occasional agitation. After 1X PBS wash, cells were incubated with 1X PBS / 5% BSA / 0.3% Triton™ X-100 for 1 hr at RT with gentle rocking. Primary antibodies were added (1/400 dilution per well) and incubated overnight at 4°C with gentle rocking. After 3 washes with 1X T-PBS (PBS + 0.1% Tween 20) for 5 min at RT, secondary antibodies were added , Alexa Fluor® 488 conjugated goat anti-Mouse IgG (H+L) Superclonal™ or goat anti-Rabbit IgG (H+L) Superclonal™ (corresponding to species of primary antibody; Thermo Fisher # A-28175 and A-27034 respectively) at a dilution of 1:1000 in PBS / 0.3% Triton™ X-100/ 5% BSA for 1 hr at RT with gentle rocking. After 3 washes with 1X T-PBS for 5 min at RT, cells were incubated for 1 hr at RT with 1 µl of Nt.CviPII-pGL (NEB, 0.5 units) in 800 µl binding buffer per well (20 mM potassium phosphate pH 7.0, 20 mM NaCl, 0.3% Triton™ X-100). In some experiments, heparin (Sigma-Aldrich # H3149) was added at different concentrations to prevent non-specific DNA binding. Plates were then washed 3 times with washing buffer (20 mM potassium phosphate buffer  pH 7.0, 500 mM NaCl, 0.3% Triton™ X-100) for 15 min at RT. Cells were incubated with 10 units of DNA Polymerase I (NEB # M0209S), 5 mM of dNTP mix including Biotin-14-dCTP (Thermo Fisher # 19518018), 5-methyl-dCTP (NEB # N0356S), dATP, dTTP and dGTP for 1 hr at 37°C in 800 µl of 1X NEBuffer 2 per well to allow nick translation to occur. Plates were then washed 3 times with washing buffer (20 mM potassium phosphate buffer, pH 7.0, 500 mM NaCl, 0.3% Triton™ X-100) for 15 min at RT with an additional wash with 1X PBS. Cells were then covered with 100 µl of 1X PBS and DNA was extracted using Monarch genomic DNA purification kit (NEB # T3010S) according to manufacturer’s recommendations. After DNA purification, 100-500 ng of genomic DNA was fragmented using 2 units of Nt.CviPII (NEB # R0626S) in 500  µl of 1X NEBuffer 2 at 37°C overnight. Nt.CviPII was inactivated at 65°C for 20 min. NaCl 5M stock was added to reach 1 M final concentration along with 30 µl of Streptavidin magnetic beads (NEB # S1420S) for 2 hrs with end-over-end mixing at 4°C to capture biotinylated DNA fragments. Streptavidin bound DNA beads were washed twice with 1X TE buffer supplemented with 2 M NaCl and 0.05 % Triton™ X-100. A third wash was performed with 1X TE buffer. Beads were resuspended in 50 µl of TE buffer and libraries were made on beads using NEBNext®Ultra™ II DNA Library Prep Kit for Illumina® (NEB # E7645S) according to manufacturer’s protocol. 8 to 12 PCR cycles were used to amplify barcoded DNA. NEED-seq protocol described above was used with some modifications. Briefly, K562 crosslinked cells with 4% formaldehyde were decrosslinked overnight at 58°C in 1X PBS. Cytoplasmic extraction was performed for 30 min with end-over-end mixing at 4°C. After 300 x g centrifugation at 4°C for 5 min, cell pellet was washed once with 1X PBS and nuclei number was calculated using Luna automated cell counter. Nuclei resuspended in 1X PBS were then captured by adding 10 µl of preactivated Concanavalin A magnetic beads (Cell Signaling Technology # 93569) for 10 min with end-over-end mixing at RT. Bead-bound nuclei were incubated with 1X PBS including 0.3 % Triton X-100 and 5% BSA for 1 hr at RT. Primary antibodies were added (1/400 final dilution) and incubated overnight with end-over-end mixing at 4°C. After 3 washes with 1X T-PBS for 5 min at RT, blocking buffer was added with anti-mouse IgG or anti-rabbit IgG coupled with Alexa 488 for 1h at RT with gentle rocking. After 3 washes with 1X T-PBS for 5 min at RT, beads were resuspended in 1 ml binding buffer (as described above) including 1 ul of fusion enzyme Nt.CviPII-pGL (NEB) for 1 hr at RT. Beads were then washed 3 times with washing buffer (as described above) for 15 min at RT. After 1 wash with 1X PBS, beads were incubated with 10 units of DNA Polymerase I, 5 uM of dNTP mix including Biotin-14-dCTP, 5-methyl-dCTP, dATP, dTTP and dGTP for 1 hr at 37°C in 800 µl of 1X NEBuffer 2 with end-over-end mixing. After 3 washes with washing buffer for 15 min at RT, beads were resuspended in 50 mM Tris-Cl, pH 7.5, 1 % SDS, 200 mM NaCl and 2 µl of proteinase K (NEB # P8107S) and incubated overnight at 65°C to lyse nuclei bound to the beads and DNA was extracted using phenol-chloroform and precipitated using glycogen. 200 ng of DNA was fragmented using Nt.CviPII and used to make libraries as described above. For low number of cells, barcoded DNA was amplified using ~18 PCR cycles. To determine the dynamic range of NEED-seq, cells were serially diluted after the labeling reaction. In this modified NEED-seq method, HT1080 cells were grown on 6 well plate to 80% confluency. Cells were incubated for 10 min at 4°C using CSK buffer (10 mM PIPES pH 6.8, 300 mM sucrose, 100 mM NaCl, 1.5 mM MgCl2 and 0.5 % Triton X-100) followed by crosslinking with 4% formaldehyde for 10 min at RT. Glycine was used to quench formaldehyde for 5 min at RT (final concentration 0.25M). After 1X PBS wash, blocking buffer was added to cells for 1h at RT (5% BSA, 1X PBS and 0.3% Triton X-100). Different antibodies (see Table S2) were then added at dilutions recommended by the manufacturer and incubated with cells for 16 hrs at 4°C. After 3 washes with T-PBS (PBS with 0.1 % Tween 20) for 5 min at RT, blocking buffer was added with anti-mouse IgG or anti-rabbit IgG coupled with Alexa 488 for 1h at RT. After 3 washes with T-PBS for 5 min at RT, NEED-seq binding buffer (20 mM KPO4, 150 mM NaCl and 0.3% TritonX-100) was added along with Nt.CviPII-pGL enzyme (1 unit per well) for 1h at 37°C. After 3 washes with NEED-seq washing buffer (20 mM KPO4, 500 mM NaCl and 1% TritonX-100) for 15 min at RT, 1X NEBuffer2.1 was added for 1h at 37°C to allow Nt.CviPII-pGL activity to fragment the targeted DNA. After 1 wash with 1X PBS, genomic DNA extraction was performed using Monarch genomic DNA purification kit (6 wells pooled per antibody) according to NEB recommendations with a minor modification which included a lysis step at 56°C for 1h instead of 10 min to reverse DNA crosslinking. 0.1 to 1 µg of genomic DNA was used for size selection using first 0.4x volume of NEBNext Sample Purification beads with 10 min incubation at RT. Supernatant containing 200-500 bp DNA sizes was separated from beads using 12-tube magnetic separation rack (NEB # S1509S) and collected. An additional 0.4x volume of NEBNext Sample Purification beads was added to the supernatant and incubated for 10 min at RT. After 2 washes with 80% EtOH, DNA was eluted form beads with 50 µl of low TE buffer and DNA libraries were prepared using NEBNext Ultra II DNA Library Prep (NEB # E7103L) with some modifications. Briefly, end repair/dA tailing was performed on size selected DNA and NEBNext Illumina adaptor (1/10 dilution) were ligated for 2 hrs at RT. USER enzyme (NEB # E7103L) was incubated for 20 min at 37°C and another round of library size selection was performed as described above. The library was amplified using 13 PCR cycles. PCR products were purified using 0.9x volume of NEBNext Sample Purification beads. 1 nM of size selected DNA library was sequenced on Novaseq 6000. HT1080 cells (0.5x10e6) were crosslinked with 4% formaldehyde for 10 min at RT and quenched with 1.5 M Tris-HCl (pH 8) for 5 min at RT. After 1X PBS wash, cells were permeabilized with Hi-C lysis buffer (10 mM Tris-HCl (pH 8), 10 mM NaCl and 0.2% NP40) for 30 min at 4°C, washed with 1X NEBuffer2 and incubated for 10 min at 58°C with 1X NEBuffer2, 0.3% SDS and 1 mM DTT. To quench SDS, Triton X-100 was added (1% final concentration per well) for 15 min at 37°C. 400 U of MboI (NEB # R0147M) were added and incubated for 16 hrs at 37°C. Enzyme was inactivated at 58°C for 20 min. Supernatant was discarded and DNA polymerase I mix containing  NEBuffer2.1 1X (NEB # B7002S), 30 µM of dNTPs (including dCTP/dGTP/dTTP (NEB # N0441S/ N0442S/N0443S respectively) with biotin-14-dATP (ThermoFisher Scientific # 19524016)) and 50 U of DNA polymerase I (NEB # M0210M) was added and incubated for 4 hrs at RT. Ligase mix was added and incubated for 16 hrs at RT. Ligase mix contains 2000 U of T4 DNA ligase (M0202M NEB), 140 µg of recombinant albumin (NEB # B9200S),  1X T4 DNA ligase buffer (NEB # B0202S) and 1 % Triton X-100. After ligation, supernatant was discarded, and cells were washed using 1X PBS. Genomic DNA was extracted from the wells using Monarch Genomic DNA purification Kit (NEB # T3010L). To remove biotin from un-ligated DNA ends, 10 µg of genomic DNA was incubated with 30 U of T4 DNA Polymerase (NEB # M0203L), 25 µM dATP/dGTP (NEB # N0440S/N0442S respectively) in 1X NEBuffer2 for 4 hrs at 20°C. Enzyme was inactivated for 20 min at 75°C. DNA was purified using 1X volume of NEBNext Sample Purification beads (NEB # E6178S) and sheared to 300 bp using Covaris (program : repeat/iterations # 7; repeat process treatment duration 10s; peak power 50W; duty factor 20%; cycles per burst 1000; total treatment time per sample 70s). To capture biotinylated DNA, DNA was incubated with NEB Magnetic Streptavidin beads (NEB # S1420S) for 30 min at RT in 10 mM Tris-HCl (pH 7.5) supplemented with 1 M NaCl. Beads were washed once with 10 mM Tris-HCl (pH 7.5) supplemented with 1 M NaCl and once with low TE buffer. Beads were resuspended in 50 µl of low TE buffer and DNA libraries were prepared on beads using NEBNext Ultra II DNA Library Prep (NEB # E7103L) with some modifications. Briefly, end repair/dA tailing was performed on beads and NEBNext Illumina adaptor (1/10 dilution) were ligated for 2 hrs at RT. 3 µl of USER (NEB # E7103L) was added for 20 min at 37°C. Beads were then washed once with 10 mM Tris-HCl (pH 7.5) supplemented with 1 M of NaCl and once with low TE. Beads were resuspended in 15 µl of low TE. 14 PCR cycles were performed. Supernatant containing PCR products were separated from beads using 12-tube magnetic separation rack (NEB # S1509S) and purified using 0.9x volume of NEBNext Sample Purification beads. 1 nM of DNA Hi-C library was loaded on Novaseq 6000.

value: Lamin B2


title: H3K27me3_MNase+37C (Size Selection)


title: PXB IRF2 E077

source_name: primary human hepatocytes derived from chimeric mice with human liver

value: HBV infection, si-IRF2

value: primary human hepatocytes


title: PXB Non E077

value: HBV infection, si-Non target


title: Rbp4,F,DOI,1month,rep2,b1

description: Library name: AG-2067

value: female


title: LaminB1

value: Lamin B1


title: WT2

city: BEIJING

first: HAO

last: wenyan

department: School of Basic Medical Sciences

institute: Peking University Health Science Center

description: Library name: Sample 4

data_processing:
Illumina Casava2.19 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to hg38 whole genome using hisat2 v2.0.5 with parameters hisat2 [options]* -x <hisat2-idx> {-1 <m1> -2 <m2> | -U <r> | --sra-acc <SRA accession number>} [-S <hit>]
Fragments Per Kilobase of exon per Megabase of library size (FPKM) were calculated using a protocol from Trapnell,Cole et al., Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol, 2010. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Assembly: mm10
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

source_name: liver

extract_protocol:
Total RNA was extracted with Trizol reagent (Invitrogen) and reverse transcribed into cDNA with GoScriptTM Reverse Transcription System (Promega). 1 ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using standard Illumina protocols.

value: liver

value: wild type

value: MHV infection


title: Meninges.IgG#1

value: Whole tissue

value: IgG (isotype control)


title: WT1

description: Library name: Sample 3


title: HepG2_si-STAT1

value: HBV transfected, si-STAT1


title: Meninges.aCD25#2

value: Meninges


title: H3K27me3_Nt.CviPII (Size Selection)

value: fibrosarcoma cells


title: HiC

description: HiC

data_processing:
Adaptor and low-quality sequences were trimmed from paired-end sequencing reads using Trim Galore v 0.5.0
NEED-seq:Trimmed read pairs were mapped to the reference genome (human: hg38) using Bowtie2 v. 2.3.4.3. PCR duplicates and mitochondrial reads were removed using Picard tools and samtools. The aligned bam files of technical replicates were merged using samtools. Open chromatin regions were identified by calling narrow peaks and Closed chromatin were identified by calling broad peaks using MACS2.
EM-seq: Trimmed read pairs were mapped to the reference genome (human: hg38) using Bwa-meth. PCR duplicates and mitochondrial reads were removed using samblaster and samtools.
ChIP-seq: Trimmed read pairs were mapped to the reference genome (human: hg38) using Bowtie2 v. 2.3.4.3. PCR duplicates and mitochondrial reads were removed using Picard tools and samtools. The aligned bam files of technical replicates were merged using samtools.  Open chromatin regions were identified by calling narrow peaks and Closed chromatin were identified by calling broad peaks using MACS2 where Input were used as control
Hi-C: HiC data were analysed using HicPro.
Assembly: hg38
Supplementary files format and content: Bigwigs were generated using bins using deeptools bamCoverage with the following parameters: --normalizeUsing RPKM

value: Chromosome Conformation


title: spermatozoa, proven-fertile, 10

description:
no duplicate
10

value: ejaculate

value: spermatozoa

value: proven-fertile


title: Microglia.DTR+#2

value: Hippocampus

value: Microglia

value: Diphtheria toxin


title: KO2

description: Library name: Sample 2

value: ZUP2 knock-out


title: H-RE-4

city: Wuhan

first: Mingliang Wei

last: Mingliang Wei

state: Hubei

address: 299 Bayi Road, Wuchang District

institute: Wuhan University

description: Library name: H-RE-4

data_processing:
Adapters and low-quality bases were removed with Fastp (version 0.23.4, https://github.com/OpenGene/fastp) using the following parameters: “-f 10 -F 10 -l 30 --adapter_sequence AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC --adapter_sequence_r2 AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT”.
The cleaned RNA-seq reads were then aligned to the mouse UCSC reference genome (mm10) using HISAT2 (version 2.2.1, https://daehwankimlab.github.io/hisat2/) with default settings.
Uniquely aligned reads were counted at gene regions using the featureCounts package (version 2.0.6), based on Gencode vM25 annotations.
Differential gene expression analysis was performed using the R package DESeq2 (version 1.40.1, https://bioconductor.org/packages/release/bioc/html/DESeq2.html).
Assembly: mm10
Supplementary files format and content: table-delimited txt file includes raw counts for each samples

source_name: Liver

extract_protocol:
RNA was harvested using RNApure Tissue&Cell Kit (CWBIO). 2ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using Fast RNA-seq Lib Pre Kit V2 (ABclonal) following manufacturer's protocols.

value: Liver

value: HFD+RE


title: PXB_si-STAT1

value: HBV infection, si-STAT1

value: primary human hepatocytes


title: H-AE-3

description: Library name: H-AE-3

value: HFD+AE


title: Microglia.DTR+#1


title: polysome_Sc-2

city: Shanghai

first: Lei

last: Hou

state: Shanghai

address: 746 Zhongshan Middle Road

department: Department of Cardiology

institute: Songjiang Hospital and Songjiang Research Institute, School of Medicine, Shanghai Jiao Tong University

description:
Library name: Sample 2
polysome_Sc-2 in FPKM_polysome.txt

library_source: other

data_processing:
Trimmed sequence reads were mapped to GrCM38/mm10 using CLC genomic benchwork (parameters - mismatch cost: 2 insertion cost: 3 deletion cost: 3 length fraction: 0.8 similarity fraction: 0.8)
Assembly: NCBI_GCF_000001635.27_GRCm39
Supplementary files format and content: tab-delimited text file includes TPM for each Sample

molecule: other

source_name: ADSCs

extract_protocol:
RNA was harvested using Trizol (Takara). 800 ug of total RNA was used for the construction of sequencing libraries.
The extracted mRNA is enriched using mRNA Capture Beads. After purification with beads, the mRNA is fragmented using high temperatures. The fragmented mRNA is then used as a template to synthesize the first strand of cDNA in a reverse transcription enzyme mixture system. While synthesizing the second strand of cDNA, end repair and A-tailing are completed. Next, adapters are ligated, and Hieff NGS® DNA Selection Beads are used for purification to select target fragments.

value: ADSCs

value: WT

value: Scrambled

value: batch 2


title: KO1

description: Library name: Sample 1

value: liver

value: ZUP1 knock-out

value: MHV infection


title: Meninges.IgG#3

value: Meninges

value: Whole tissue

value: IgG (isotype control)


title: H-RE-5

description: Library name: H-RE-5


title: Meninges.IgG#2


title: polysome_Kd-2

description:
Library name: Sample 4
polysome_Kd-2 in FPKM_polysome.txt

value: IFIT1 knockdown


title: Microglia.DTR-#2

value: Hippocampus

value: Microglia

value: Diphtheria toxin


title: EMseq_rep3

data_processing:
Library Strategy: EM-seq
Adaptor and low-quality sequences were trimmed from paired-end sequencing reads using Trim Galore v 0.5.0
NEED-seq:Trimmed read pairs were mapped to the reference genome (human: hg38) using Bowtie2 v. 2.3.4.3. PCR duplicates and mitochondrial reads were removed using Picard tools and samtools. The aligned bam files of technical replicates were merged using samtools. Open chromatin regions were identified by calling narrow peaks and Closed chromatin were identified by calling broad peaks using MACS2.
EM-seq: Trimmed read pairs were mapped to the reference genome (human: hg38) using Bwa-meth. PCR duplicates and mitochondrial reads were removed using samblaster and samtools.
ChIP-seq: Trimmed read pairs were mapped to the reference genome (human: hg38) using Bowtie2 v. 2.3.4.3. PCR duplicates and mitochondrial reads were removed using Picard tools and samtools. The aligned bam files of technical replicates were merged using samtools.  Open chromatin regions were identified by calling narrow peaks and Closed chromatin were identified by calling broad peaks using MACS2 where Input were used as control
Hi-C: HiC data were analysed using HicPro.
Assembly: hg38
Supplementary files format and content: Bigwigs were generated using bins using deeptools bamCoverage with the following parameters: --normalizeUsing RPKM

value: fibrosarcoma cells

value: Methylation


title: Microglia.DTR-#1


title: H3K27me3_SizeSelection


title: polysome_Kd-1

description:
Library name: Sample 3
polysome_Kd-1 in FPKM_polysome.txt

value: batch 1


title: H-HFD-3

description: Library name: H-HFD-3

value: Liver

value: HFD


title: H-RE-3

description: Library name: H-RE-3

value: HFD+RE


title: EMseq_rep2


title: skull.BM#3

source_name: Skull bone marrow

value: Skull bone marrow

value: Treg cell


title: EMseq_rep1


title: PXB_si-Non Target

value: HBV infection, si-Non target

value: primary human hepatocytes


title: skull.BM#2


title: polysome_Sc-1

description:
Library name: Sample 1
polysome_Sc-1 in FPKM_polysome.txt

value: ADSCs

value: WT

value: Scrambled


title: Microglia.DTR-#3


title: IgG_sizSelection

value: Input


title: skull.BM#1


title: TR-KD-3

description:
Library name: Sample 6
TR-KD-3 in FPKM_shifit1ADSC.txt

value: IFIT1 knockdown

value: batch 3


title: H-AE-2

description: Library name: H-AE-2

value: HFD+AE


title: TR-KD-2

description:
Library name: Sample 5
TR-KD-2 in FPKM_shifit1ADSC.txt

value: batch 2


title: H-AE-1

description: Library name: H-AE-1


title: H2AK119ub_SizeSelection

value: fibrosarcoma cells


title: H3K4me3+ChIP

value: Chronic myelogenous leukemia cells


title: TR-KD-1

description:
Library name: Sample 4
TR-KD-1 in FPKM_shifit1ADSC.txt

value: batch 1


title: H3K9me2_SizeSelection


title: H-NC-4

description: Library name: H-NC-4

value: Liver

value: NC


title: TR-Sc-3

description:
Library name: Sample 3
TR-Sc-3 in FPKM_shifit1ADSC.txt


title: H-HFD-2

description: Library name: H-HFD-2

value: HFD


title: H3K9me3_SizeSelection


title: H3K27me3+ChIP


title: H-HFD-1

description: Library name: H-HFD-1


title: Treg.1year#2

value: Meninges

value: Treg cell


title: VEH_01 [S448.01]

data_processing:
The samples were sequenced using a 2x150bp Paired End (PE) configuration.
Sequence reads were trimmed using Trimmomatic v0.36 and mapped using STAR aligner v.2.5.2b.
Unique gene hit counts were calculated by using featureCounts from the Subread package v.1.5.2.
Using DESeq2, a comparison of gene expression between the customer-defined groups of samples was performed. The Wald test was used to generate p-values and log2 fold changes. Genes with an adjusted p-value < 0.05 and absolute log2 fold change > 1 were called as differentially expressed genes for each comparison.
The Voom Normalize module in GenePattern was used to normalize the entire dataset for downstream analysis in BMDExpress2.
Assembly: mRatBN7.2
Supplementary files format and content: tab-delimited text files with read counts

organism: Rattus norvegicus

source_name: fetal testis

growth_protocol: Rat fetal testes obtained from timed pregnant Sprague Dawley rats on gestational day (GD) 21.

extract_protocol:
Total RNA was extracted using the Zymo Research Quick-RNA MicroPrep Kit per manufacturer's instructions, including DNase treatment. 1-2 testes from fetuses in the same litter were pooled for each RNA sample.
The RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturer’s instructions (New England Biolabs, Ipswich, MA, USA), with polyA selection for mRNA enrichment.

treatment_protocol: Timed pregnant rats were treated with dipentyl phthalate (DPeP) at doses of 1, 11, 100, or 300 mg/kg/d, or vehicle control (corn oil), a 2.5 mL/kg solution of corn oil, by oral gavage.

value: fetal testis

tag: strain

value: Sprague Dawley

value: wild type

value: corn oil


title: PAT_SON_2

city: Nijmegen

first: Dyah

middle: Winiarty

last: Karjosukarso

address: Geert Grooteplein Zuid 10

country: Netherlands

institute: Radboudumc

data_processing:
Sequence reads were trimmed to remove possible adapter sequences and filtered on low-quality bases using fastp v0.23.2
The trimmed reads were mapped to the human GRCh38.p13  (Homo_sapiens.GRCh38.dna.primary_assembly.fa) reference provided in the References directory. The reads were mapped to the reference sequence using a short read aligner based on Burrows-Wheeler Transform (STAR2 v2.7.10) with default settings.
Based on the mapped locations in the alignment file the frequency of how often a read was mapped on a transcript was determined with HTSeq v2.0.2. T
Differential expression analysis was performed using DESeq2 v1.39.8)
Differential splicing analysis was carried out by utilizing Leafcutter v0.2.9.
Assembly: hg38
Supplementary files format and content: tab delimited file of counts

source_name: retinal organoids

extract_protocol:
Total RNA was isolated using Nucleospin RNA Mini kit (Macherey Nagel) following manufacturer’s instructions.
The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina was used to process the sample(s). The  sample preparation was performed according to the protocol "NEBNext Ultra II Directional RNA Library Prep  Kit for Illumina" (NEB #E7760S/L). Briefly, rRNA was depleted from total RNA using the Qiagen fast select kit  (#334376). After fragmentation of the rRNA reduced RNA, a cDNA synthesis was performed. This was used for  ligation with the sequencing adapters and PCR amplicification of the resulting product. The quality and yield  after sample preparation was measured with the Fragment Analyzer (see appendices). The size of the  resulting products was consistent with the expected size distribution (a broad peak between 300-500 bp).

value: retinal organoids

value: ABCA4 c.768T

value: SON


title: TR-Sc-2

description:
Library name: Sample 2
TR-Sc-2 in FPKM_shifit1ADSC.txt

value: ADSCs

value: WT

value: Scrambled


title: Input

description: Input

value: Chronic myelogenous leukemia cells

value: Input


title: Treg.1year#1

data_processing:
Reads were mapped to the mouse reference genome (mm10) with STAR 2.7.3a
Non-uniquely mapped reads were discarded.
The uniquely mapped reads were assigned to genes using featureCounts with GENCODE annotation (vM25)
Assembly: mm10
Supplementary files format and content: tab-delimited text files include raw read counts for each Sample

source_name: Meninges

tag: tissue


title: H-NC-5

description: Library name: H-NC-5

value: Liver


title: RMS_Snord89_Ctrl_A

data_processing:
Adapter trimming was performed using either trim_galore or cutadapt.
Mapping was performed using bowtie2 with --end-to-end -k 1 options.
Mapped bam files were converted to bed using bedtools bamtobed then 5' and 3' end counting was performed using "awk '{print $2}' RMS_${sample}.bed | sort | uniq -c | awk '{print $2, $1}' | sort -n > RMS_${sample}.5count.txt" and "awk '{print $3}' RMS_${sample}.bed | sort | uniq -c | awk '{print $2, $1}' | sort -n > {.}.3count.txt"
RMS meth score was calculated considering weighted 5' or 5'+3' counts of neighboring 12 nucleotides as described in Birkedal et al. 2014.
Assembly: Reads were mapped to 45S rRNA (NR_046233.2) and U2 snRNA (NR_004414.1).
Supplementary files format and content: Tab delimited text file containing end counts (5' only or 5' and 3') and the calculated RMS score at each nucleotide position in 45S rRNA or U2 snRNA for each of the replicate RMS libraries. Mean RMS scores are also provided.
Library strategy: RiboMeth-seq

source_name: ESC

extract_protocol:
Total RNA was extracted using Trizol.
150 to 250 ng of DNase-treated RNA was hydrolyzed in 50 mM sodium bicarbonate buffer (pH 9.2) for 1 to 12 minutes. The hydrolysis reaction was quenched in 1 mL EtOH containing 10 uL 3 M NaOAc, pH 5.2 (Thermo) and 15 ug Glycoblue coprecipitant (Thermo) and submerged in liquid nitrogen. Hydrolyzed RNA was then precipitated and washed in 80% EtOH. The intended fragment size of around 50-100 bp was verified on the TapeStation 4200 system (Agilent). RNA was dephosphorylated by Antarctic Phosphatase (New England Biolabs) for 30 minutes at 37°C, and end-repaired by T4 PNK (New England Biolabs) in the presence of 1 mM ATP (New England Biolabs) for 1 hour at 37°C. End-repaired RNA was purified using RNA Clean & Concentrator-5 columns (Zymo). Illumina libraries were then prepared using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® kit (New England Biolabs) and purified on NucleoSpin® Gel and PCR Clean-up columns (Takara Bio).

value: ESC

tag: cell type

value: iNeuron

value: non-targeting ASO


title: Treg.16wks#2

value: Meninges

value: Treg cell


title: Meninges#2


title: DPeP300_06 [S448.06]

value: fetal testis

value: Sprague Dawley

value: 300 mg/kg/d DPeP


title: Treg.16wks#1


title: DPeP300_14 [S448.14]

value: wild type


title: RMS_Snord89_KD_B

description: RiboMeth-seq from mouse induced neurons treated with Snord89-targeting ASO, biological replicate 2

value: Snord89 ASO


title: Meninges#1


title: Female.Tregs#2


title: H-NC-2

description: Library name: H-NC-2

value: NC


title: DPeP11_16 [S448.16]

value: 11 mg/kg/d DPeP


title: DPeP100_18 [S448.18]

value: 100 mg/kg/d DPeP


title: Male.Tregs#2


title: DPeP300_20 [S448.20]


title: RMS_Snord89_KD_A

description: RiboMeth-seq from mouse induced neurons treated with Snord89-targeting ASO, biological replicate 1


title: ISO_AON_1

value: retinal organoids

value: AON


title: Male.Tregs#1


title: DPeP11_03 [S447.03]


title: RMS_Snord89_Ctrl_B

description: RiboMeth-seq from mouse induced neurons treated with non-targeting ASO, biological replicate 2


title: PAT_AON_2

value: ABCA4 c.768T


title: DPeP100_05 [S447.05]


title: DPeP1_02 [S448.02]

value: 1 mg/kg/d DPeP


title: flow

city: Neuherberg

first: Sandra

last: Wiedenmann

state: Bavaria

address: Ingolstädter Landstraße 1

department: Helmholtz Pioneer Campus

country: Germany

institute: Helmholtz Zentrum München

library_source: transcriptomic single cell

data_processing:
Raw sequencing data files were demultiplexed, aligned (reference genome hg38_ensrel96), filtered, barcodes and UMIs counted, and subjected to a quality filter with CellRanger (10xGenomics).
The pre-processing and downstream analysis were performed with the package ‘Scanpy62 API’88  (version 1.9.1) in python with default parameters, if not stated differently
For velocity analysis, alignment files (bam) were analyized by samtools ( version 1.16) and then "velocyto run" command was applied on sorted barcodes in Cell Ranger. The generated loom files were then analyzed together with the filtered,normalized and cluster annotated scRNAseq analysis file with the basic anaylsis pipeline of scvelo package(version  0.2.5)
Assembly: hg38_ensrel96
Supplementary files format and content: compressed matrix files

source_name: hiPSCs

growth_protocol: HiPSCs (HMGUi001-A) were differentiated to endothelial cells in a 3D shaking culture approach and sorted for CD31+ cells on day 6 as described before. SC-ECs were cultured on fibronectin-coated plates (Sigma, 5 µg/ml final concentration) in StemPro-34 (LIFE Technologies), with 15% FBS, VEGFA and FGF2 (100 ng/ml each) with medium change every other day

extract_protocol:
For RNA single cell sequencing, chips were washed with PBS. To ensure that only cells growing in the hydrogel were collected, inlet and outlet ports were blocked with 3% agarose. After solidification of the agarose, cells were treated with a 1:1 mix of Collagenase P (18 U final concentration, Roche 11213857001) and Accutase and incubated for 30 min at 37°C. Pipette tips and tubes were washed with PBS + 2% BSA before usage to reduce cell loss. Successfully dissolved hydrogels and a single cell suspension was confirmed under the microscope before harvesting the cells in PBS + 10% FBS. The cells were centrifuged at 300 x g for 3 min, washed with PBS + 2% BSA and filtered with a 20 µm cell Pluristrainer (pluriSelect, 43-50020-023). Cells were counted with TrypanBlue (Sigma) and a cell vitality of over 70% was confirmed.
RNA libraries were generated using Chromium Single Cell 3’ library and gel bead kit v3.1 (10x Genomics).
The amplified cDNA library was sequenced on a NovaSeq SP flow cell from Illumina.

treatment_protocol: Endothelial cells were seeded into a pre-molded lumen which is embedded in a Collagen I hydrogel environmenton an microfluidic Vesssel-on-Chip platform. Either lumen were perfused by gravity driven flow on a shaker (25°, 2 min interval) or left in static condition for total 3 days. After seeding, EGM-2 (Sigma Aldrich, C-22011) medium was added to all chambers. Media change was performed every day.

value: hiPSCs

value: endothelial

value: wt

value: flow


title: TR-Sc-1

description:
Library name: Sample 1
TR-Sc-1 in FPKM_shifit1ADSC.txt

value: ADSCs

value: WT

value: Scrambled

value: batch 1