title: miR-146a-/- ESLAM_TGGGGTT

city: Vancouver

first: Aly

last: Karsan

state: British Columbia

address: 675 W 10th Ave

institute: BC Genome Science Centre

description:
C8AP5ANXX_1_1_TGGGGTT_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice

data_processing:
Basecalls performed using Illumina's CASAVA software, version 1.8.2
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm8 whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
The first 6 bases were trimmed using Trimgalore v0.4.0 and Cutadapt v1.2.1 using the parameters --clip_R1 6 --clip_R2 6 –paired. Trimmed fastq files were aligned using Novoalign version V3.02.10 (www.novocraft.com) to the mouse assembly GRCm38 (mm10). Alignments were done in paired-end mode using the options -b4 (non-directional), -t 20,3 (optimized for SNP concordance), –a (adapter trimming), --hlimit 8 (homopolymer filter), and -H 20 (removes trailing bases with quality ≤ 20), and –u8 (penalty for unconverted CHG or CHH cytosine) as recommended by Novoalign. Aligned reads were output into the BAM format and sorted using SAMtools V0.1.17. Duplicates were marked with Picard V1.31 (http://picard.sourceforge.net).
Methylation of each cytosine was called using SAMtools mpileup (-B -C 0 -q 30 -d 500) followed by Novomethyl V1.01 (-o Consensus -%) (www.novocraft.com). Cytosine calls with Phred quality score ≤15 were discarded. Methylation percentage of each CpG di-nucleotide (found by searching the genome with a custom java script) was calculated by taking a weighted average of each cytosine using the map command from the bedtools suite (Quinlan and Hall, 2010). In most cases, only one base within a CpG dinucleotide had coverage; in these cases, the methylation information of the covered base was extrapolated to the other base.
Genome_build: mm10
Supplementary_files_format_and_content: bed file

source_name: miR-146a-/- ESLAM

extract_protocol:
Sorted cells were lysed in 96 well plates in 4 uL of lysis buffer (20 mM Tris-HCl, pH 8.0, 20 mM KCl, 0.3% Triton-X 100, 1 mg/mL Qiagen Protease) for 30 minutes at 50 degrees.
The lysed cells were then spiked-in with 1 uL of 60 fg/uL purified T7 phage DNA (GeneON). The DNA mixture was then bisulfite converted with the MethylEdge Bisulfite Conversion kit (Promega) using a modified bead-based protocol which enables automation (Domanico et al., 2013): 5 uL of the lysis/spike-in mixture was combined with 32.5 uL of MethylEdge Conversion reagent and incubated at 98°C for 8 minutes and 54°C for 60 minutes in a PCR machine. After incubation, the plate was briefly spun down and placed on an Agilent Bravo robot for subsequent steps. The 37.5 uL of bisulfite converted DNA was mixed with 180 uL of MethylEdge Binding Buffer and 1.8 uL of 20 mg/mL MagSi-DNA allround magnetic silica beads (MagnaMedics) and incubated at room temperature for 15 minutes. The beads were then collected on a magnet for 3 minutes and washed twice with 220 uL of 80% ethanol for 30 seconds while on the magnet. 60 uL of MethylEdge desulfonation buffer was added and incubated for 15 minutes. The desulfonation buffer was then removed and the beads were washed twice with 100 uL of 80% ethanol. The beads were then air-dried for 3 minutes, resuspended in 20 uL of Qiagen elution buffer, and incubated at 56°C in a shaking incubator at 2,000 rpm for 15 minutes. The beads were then collected on a magnet for 30 seconds and the supernatant containing the DNA was removed. The bisulfite-converted ssDNA from above was added directly into a mixture containing 1.25 uL of 10 mM dNTPs and 1 uL of 500 uM random hexamers (3’ phosphothioate). The reactions were incubated at 98°C for 1 minute and snap frozen on ice for 2 minutes. A mastermix of 0.5 uL of 50 U/uL Klenow fragment [3’→5’ exo-] (NEB M0212M), and 2.5 uL of 10X NEB Buffer 2 was added on ice and mixed well. The reactions incubated in a PCR machine as follows: 4˚C for 10 minutes, ramped from 4°C to 37°C at 4°C/s, and held at 37°C for 30 minutes. The entire reaction was denatured again at 98˚C for 1 minute and snap frozen on ice for 2 minutes. The reaction was transferred on a pre-chilled CoolRack by robot into a new plate containing 5 uL mixture of 20 uM random hexamers (3’ phosphothioate), 0.5 mM dNTPs, 1X NEB buffer and 25 U of Klenow fragment [3’→5’ exo-]. The wells were mixed well and the reaction was cycled once again: 4˚C for 10 minutes, ramped from 4°C to 37°C at 4°C/s, and held at 37°C for 30 minutes, and heat-inactivated at 70°C for 10 minutes. 20 uL of Qiagen Elution Buffer was added to each reaction to make it up to 50 uL, and each reaction was purified using 50 uL of Sera-Mag beads before proceeding to the standard illumina adapter ligation library construction protocol. The 25 uL of cleaned-up adapter-ligated fragments were PCR amplified in 50 uL containing 1 U of Phusion U Hot Start DNA Polymerase (Life Technologies F-555S), 1X HF buffer, 3% DMSO, 200 uM dNTPs, 0.5 uM PCR primer 1.0 (need sequence) (IDT), and corresponding 0.5 uM indexing PCR primer 2.0. The reactions were incubated in a PCR machine as follows: initial 98˚C for 1 minute; 10 cycles of 98˚C for 30 seconds, 65˚C for 15 seconds, 72˚C for 15 seconds; and a final extension of 72˚C for 5 minutes. The 50 uL reaction was cleaned up with 40 uL of Sera-Mag beads and eluted in 15 uL.

tag: strain background

value: 10-14 weeks

value: bone marrow

tag: cells

value: ESLAM (EPCR+ CD45+ CD150+ CD48-)

tag: molecule subtype

value: Post-Bisulfite Adapter Ligation DNA


title: miR-146a-/- ESLAM_TTAATAT

status: Public on May 14 2020

type: SRA

country: Canada

description:
C8AP5ANXX_1_1_TTAATAT_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice

library_source: genomic

molecule: genomic DNA

organism: Mus musculus

tag: genotype

tag: age

tag: tissue


title: miR-146a-/- ESLAM_TTGACCA

description:
C8AP5ANXX_1_1_TTGACCA_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_TTCGCCT

description:
C8AP5ANXX_1_1_TTCGCCT_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_TTTAGGC

description:
C8AP5ANXX_1_1_TTTAGGC_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_TGCACTT

description:
C8AP5ANXX_1_1_TGCACTT_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_TGACTAG

description:
C8AP5ANXX_1_1_TGACTAG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_TGCAAGG

description:
C8AP5ANXX_1_1_TGCAAGG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_TGCTCCA

description:
C8AP5ANXX_1_1_TGCTCCA_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: QXT73

city: Bethesda

first: Gernot

last: Wolf

state: Maryland

address: 6 Center Drive

department: NICHD

country: USA

institute: NIH

data_processing:
Library strategy: gDNA capture-seq
R1 and R2 reads were mapped to the mm9 genome separately, each both in under settings that only allows non-duplicated, uniquely mappable reads (Bowtie -m 1 --best --strata; samtools rmdup -s) and under settings that allows multimapping and duplicated reads (Bowtie --best).
Of the multimapping reads, only reads that overlap (min. 50% of read) with RLTRETN, MMETn-int, ETnERV-int, ETnERV2-int or ETnERV3-int repeats were kept.
Non-duplicated and uniquely mappable reads whose paired read partners were overlapping ETn repeats were then clustered (as bed files) using bedtools (bedtools merge -i -n -d 1000) to receive a list of all potential annotated and non-annotated new ETn insertions.
Genome_build: mm9
Supplementary_files_format_and_content: bed files with clustered ETn-paired reads

source_name: tail

extract_protocol:
Genomic DNA was purified from Proteinase K digested lysates using the gDNA Clean & Concentrator kit (Zymo)
Libraries were constructed and enriched using the SureSelectQXT Target Enrichment kit (Agilent) and custom RNA capture probes complementary to ETn LTRs.

value: tail

tag: mouse id

tag: Sex

value: F

tag: strain

value: Chr4-cl KO/WT

tag: ancestry


title: miR-146a-/- ESLAM_TCGATGT

description:
C8AP5ANXX_1_1_TCGATGT_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_TATCACG

description:
C8AP5ANXX_1_1_TATCACG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_TGATGCT

description:
C8AP5ANXX_1_1_TGATGCT_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_TATCTAT

description:
C8AP5ANXX_1_1_TATCTAT_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_TAATGAA

description:
C8AP5ANXX_1_1_TAATGAA_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_TATACGG

description:
C8AP5ANXX_1_1_TATACGG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_TCAGATC

description:
C8AP5ANXX_1_1_TCAGATC_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_TAGCCGC

description:
C8AP5ANXX_1_1_TAGCCGC_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_GTAATCG

description:
C8AP5ANXX_1_1_GTAATCG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_TACAGTG

description:
C8AP5ANXX_1_1_TACAGTG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_GGGCTAC

description:
C8AP5ANXX_1_1_GGGCTAC_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_GCAGGCG

description:
C8AP5ANXX_1_1_GCAGGCG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_TAGGCCG

description:
C8AP5ANXX_1_1_TAGGCCG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_GTATAAT

description:
C8AP5ANXX_1_1_GTATAAT_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_GTAGCTT

description:
C8AP5ANXX_1_1_GTAGCTT_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_GCACGAT

description:
C8AP5ANXX_1_1_GCACGAT_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_GCACTCA

description:
C8AP5ANXX_1_1_GCACTCA_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_TGCCAAT

description:
C8AP5ANXX_1_1_TGCCAAT_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_GCTTGTA

description:
C8AP5ANXX_1_1_GCTTGTA_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_GCACCGG

description:
C8AP5ANXX_1_1_GCACCGG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: QXT75


title: miR-146a-/- ESLAM_GGATCAG

description:
C8AP5ANXX_1_1_GGATCAG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_GCAAAAG

description:
C8AP5ANXX_1_1_GCAAAAG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_GATGGGT

description:
C8AP5ANXX_1_1_GATGGGT_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_GAGTTTG

description:
C8AP5ANXX_1_1_GAGTTTG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: QXT67

value: Chr4-cl KO/KO


title: miR-146a-/- ESLAM_TATAATT

description:
C8AP5ANXX_1_1_TATAATT_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_TTATTTG

description:
C8AP5ANXX_1_1_TTATTTG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_GCGCATA

description:
C8AP5ANXX_1_1_GCGCATA_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_GAGGTAA

description:
C8AP5ANXX_1_1_GAGGTAA_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_CTCAGCC

description:
C8AP5ANXX_1_1_CTCAGCC_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_GAAAGCA

description:
C8AP5ANXX_1_1_GAAAGCA_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_CCGCCGA

description:
C8AP5ANXX_1_1_CCGCCGA_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_CGAGTGG

description:
C8AP5ANXX_1_1_CGAGTGG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_CCCTTAG

description:
C8AP5ANXX_1_1_CCCTTAG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_GAAACAT

description:
C8AP5ANXX_1_1_GAAACAT_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_GAAATGC

description:
C8AP5ANXX_1_1_GAAATGC_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_CCCCATG

description:
C8AP5ANXX_1_1_CCCCATG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_CAACTTG

description:
C8AP5ANXX_1_1_CAACTTG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_CAAGACT

description:
C8AP5ANXX_1_1_CAAGACT_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_CCCGCAA

description:
C8AP5ANXX_1_1_CCCGCAA_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_GCAACTA

description:
C8AP5ANXX_1_1_GCAACTA_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_CAAGGAC

description:
C8AP5ANXX_1_1_CAAGGAC_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_ATAGGAG

description:
C8AP5ANXX_1_1_ATAGGAG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_ATACCCG

description:
C8AP5ANXX_1_1_ATACCCG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_ACTCAGA

description:
C8AP5ANXX_1_1_ACTCAGA_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_ATATTGT

description:
C8AP5ANXX_1_1_ATATTGT_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_ACTATAC

description:
C8AP5ANXX_1_1_ACTATAC_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_ACTAGCT

description:
C8AP5ANXX_1_1_ACTAGCT_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_ACGTAGC

description:
C8AP5ANXX_1_1_ACGTAGC_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_AATACCC

description:
C8AP5ANXX_1_1_AATACCC_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_AATTCGA

description:
C8AP5ANXX_1_1_AATTCGA_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_AGAATAA

description:
C8AP5ANXX_1_1_AGAATAA_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_CTTGTTG

description:
C8AP5ANXX_1_1_CTTGTTG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_CACAAAC

description:
C8AP5ANXX_1_1_CACAAAC_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_AAGATAG

description:
C8AP5ANXX_1_1_AAGATAG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_AAGAAGA

description:
C8AP5ANXX_1_1_AAGAAGA_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: miR-146a-/- ESLAM_AACTCTC

description:
C8AP5ANXX_1_1_AACTCTC_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: E14 cells, iPCR_2

city: Scarborough

first: Guillaume

middle: J

last: Filion

state: Ontario

address: 1265 Military Trail

department: Biological Sciences

institute: UTSC

description: inverse PCR

data_processing:
[Processing inverse PCR reads]  The paired-end reads were pre-processed by custom Python scripts. The forward read consists of the barcode, a fixed 20 nucleotide watermark sequence used for identification, the NlaIII restriction site and some mouse genome sequence ligated to the NlaIII site. The reverse read consists of the last 25 nucleotides of pT2 and the mouse genome sequence at the insertion site of the transposon. We used seeq version 1.1 (https://github.com/ezorita/seeq) allowing up to three errors (mismatches and indels) to identify the watermark and isolate the barcode sequence. Reads were discarded if the watermark was not found or if the barcode was not between 14 and 24 nucleotides long. We removed the first 25 nucleotides of the reverse read and cut the sequence at the first NlaIII site, if any. The reads for which this sequence was shorter than 20 nucleotides were discarded. The genomic sequences thus obtained were mapped in the mouse genome release mm9 using the GEM mapper build 1.376 (Marco-Sola et al. 2012) with options `-q ignore --unique-mapping`. The reason for not using the more recent mouse release mm10 was that the chromatin from Juan et al. (2016) were only available in release mm9. After mapping, the barcodes were clustered using Starcode (Zorita, Cuscó, and Filion 2015) allowing up to two errors (options `-d2 --print-clusters`). This removes potential sequencing errors and consolidates the barcode sequences.   The barcodes were assigned to a genomic location using custom Python scripts. Unmapped genomic sequences were discarded and sequences mapping to multiple locations were flagged as such. For each barcode, we collected all the insertion sites that totalled at least 10% of the reads and we computed their diameter, equal to the maximum of their pairwise genomic distances (infinite for two sites on different chromosomes or if one of them maps to multiple locations). If the diameter was greater than 30 nucleotides, the barcode was discarded for being used in reporters mapping to different locations. Otherwise, the barcode was kept and its location was attributed to the most frequent insertion site (they are usually within 1-2 nucleotides of each other because of small mapping artefacts).
[Processing UMI-amplicon reads]  Paired-end reads were preprocessed using custom Python scripts. The forward read consists of the barcode, the watermark sequence and the right half of the second F segment . The reverse read consists of the UMI and the left half of the first F segment. Both reads extend the mid point of the F segment by three nucleotides. If the reporter is uncut or repaired by NHEJ, forward and reverse reads do not overlap. If the reporter is repaired by SSA, forward and reverse reads overlap because there is only one F segment. We can thus isolate the reads from reporters that went through SSA by ensuring that the nucleotides in the mismatch position are reverse-complement of each other in the forward and reverse reads. Thus, we used seeq with up to 10 errors to identify the half F segments and isolate the nucleotides in mismatch position on the forward and reverse reads, together with the barcode and the UMI. Barcodes and UMIs were clustered with Starcode allowing up to 2 errors (options `-d2 --print-clusters`) and the repair events were quantified for each barcode. The barcode–UMI pairs with only one read per run were discarded. After this operation, UMIs that were associated with more than one barcode were discarded. The remaining UMIs were classified as NHEJ or SSA as explained above, and those classified as SSA were further split into A/T or G/C. This provided for each barcode the full list of events reported by UMIs. Barcodes with more UMIs reporting NHEJ than SSA and those with only one UMI were removed. Barcodes that passed all these criteria in the control experiments without I-SceI induction were removed. The global proportion of remaining UMIs reporting A/T versus G/C was used a measure of repair bias.
Genome_build: mm9
Supplementary_files_format_and_content: mapped_repair.txt.gz

source_name: ES cells

growth_protocol: mESCs were grown at 37 °C under a 95% air and 5% CO2 atmosphere on gelatin in serum/LIF medium composed of GMEM (Sigma, G5154) supplemented with 15% FBS (HyClone™), 1X MEM Non-Essential Amino Acids (Gibco, 11140 - 050), 1X GlutaMax (Gibco, 35050 - 061), 1mM Sodium Pyruvate (Gibco, 11360-070), 0.1 mM 2-Mercaptoethanol (Thermo Fisher, 31350-010) and 1,000 U/ml LIF ESGRO® (Merck Millipore, ESG1106). mESCs were passaged every 2 d with a 1:8 dilution. Cells were tested yearly for mycoplasma contamination.

extract_protocol:
Genomic DNA from transfected cells was extracted using DNeasy Blood and Tissue Kit (Qiagen, #69504)
Inverse PCR: 10 μg genomic DNA from transfected mESCs were digested using 10 μl 10 U/μL NlaIII (NEB, #R0125S) in a 50 μL final volume for 3 hours at 37 °C. The reaction was heat-inactivated at 65 °C for 20 minutes. The reaction was diluted to a final volume of 1.8 mL in 1X T4 ligase buffer (Thermo Fisher Scientific, #EL0013) to favor self-ligation events, and ligation was carried out with 600 U of T4 ligase (Thermo Fisher Scientific, #EL0013) at 16°C overnight. After ligation, samples were precipitated by ethanol, pellets were resuspended in water and column-purified (QIAGEN, QIAquick PCR purification kit #28104) eluting with 100 uL EB.  Non circularized templates were eliminated by 2 h digestion at 37°C with Plasmid-safe DNAse (Epicentre, #E3101K), the enzyme was inactivated by heating for 30 minutes at 70°C. The product was column-purified (QIAGEN, QIAquick PCR purification kit #28104). The backbone of the TRIP reporters contains a I-CeuI site outside the transposable cassette, taking advantage of this, non integrated plasmids were cut by 2 h digestion at 37 °C with I-CeuI restriction enzyme (NEB, R0699S) in a total volume of 70 ul  followed by 20 minutes heat inactivation at 65 °C. All enzymatic reactions were carried out in the recommended manufacturer's buffer. For the first round of nested PCR, 10μL of I-CeuI-digestion products was mixed in 50 μL standard Phusion polymerase reaction mix (Thermo Fisher Scientific, F530S) in GC buffer, with 0.1 μM primers IR1,2 (annealing to IR/DR sequence). The cycling conditions were as follows: 98 °C for 5 min; 98°C for 20 s, 60°C for 1 min and 72°C for 5 min (1 cycle); 98°C for 20 s, 60°C for 1 min and 72°C for 2 min (20 cycles); and 72°C for 5 min. Products of the reaction were purified using Agencourt AMPure XP beads (Beckman Coulter, A63880), and eluted in 40 μL of water . For the second round of nested PCR, 37 μL of the products was diluted to 50 μL of standard Phusion polymerase reaction mix in GC buffer with 0.1 μM primer M1 (annealing to the Illumina PE1.0 primer) and one of indexing primers M2,3. The cycling conditions were as follows: 98°C for 2 min; 98°C for 20 s, 60°C for 1 min and 72°C for 1 min (10 cycles); and 72°C for 5 min. Primers M2,3 added the Illumina PE2.0 primer and one of indices to the amplicons. Products of the reaction were purified using Agencourt AMPure XP beads (Beckman Coulter, A63880), and eluted in 40 μL of water. PCR products ran as a smear on agarose gel. The smears were specific, because they failed to appear when the mESCs were not transfected. To eliminate I-SceI sites that were not cut during DSB induction and limit the size of PCR extension, 2 μg genomic DNA from mESCs (both, with DSB induction and control without the induction) were digested using 1 μl 100 U/μL XbaI (NEB, #R0145T) and 4 μl 5 U/μL I-SceI (NEB, #R0694L) in a 50 μL final volume for 3 hours at 37 °C. Digested DNA was column-purified (QIAGEN, QIAquick PCR purification kit #28104).
UMI-amplicons: To eliminate I-SceI sites that were not cut during DSB induction and limit the size of PCR extension, 2 μg genomic DNA from mESCs (both, with DSB induction and control without the induction) were digested using 1 μl 100 U/μL XbaI (NEB, #R0145T) and 4 μl 5 U/μL I-SceI (NEB, #R0694L) in a 50 μL final volume for 3 hours at 37 °C. Digested DNA was column-purified (QIAGEN, QIAquick PCR purification kit #28104).  UMI-LA 500 ng of genomic DNA obtained after I-SceI/XbaI digestion was used as a template in a 50 μL of Q5 DNA polymerase reaction mix (NEB, M0491S), using 50 nM of a UMI-containing primer U1 in the following cycling conditions: 98°C for 4 min; 98°C for 30s, 60°C for 1 min and 72°C for 1 min (50 cycles); and 72°C for 5 min. Products of linear amplification were purified using Agencourt AM Pure XP beads (Beckman Coulter, A63880), and eluted in 40 μL of water. Linear amplification was repeated 4 times for every sample to account for the technical variability.
UMI-PCR: 500 ng of genomic DNA obtained after I-SceI/XbaI digestion was used as a template in a 50 μL of Q5 DNA polymerase reaction mix (NEB, M0491S), using 50 nM of primers U1 and U2 in the following cycling conditions: 98°C for 1 min; 98°C for 20s, 60°C for 1 min and 72°C for 4 min (6 cycles); and 72°C for 10 min. Products of UMI-PCR were purified using Agencourt AM Pure XP beads (Beckman Coulter, A63880), and eluted in 40 μL of water. UMI-PCR was repeated 4 times for every sample to account for the technical variability.

treatment_protocol: To integrate the construct into the genome of mouse ES cells, 1 million cells in 6-well plates were transfected with 2 μg of plasmid pT2_FF together with 2 μg of plasmid pCMV(CAT)T7-SB100x and 2 μg of plasmid pcDNA3.1-mCherry using Lipofectamine 2000 (Thermo Fisher, #11668027). After 24 h, mCherry-positive cells were FACS sorted. Pools of 20,000 cells were plated on 24-well plates and grown for two weeks, transferring to 100 mm dishes when the cultures reached a density of 5x106 cells/mL. Two independent cell pools of 20,000 cells were prepared for each construct. To generate the mismatches, pools of mouse ES cells harboring integrated transposons were transfected with 5 μg of plasmid pCBASceI using Lipofectamine 2000 (Thermo Fisher, #11668027). The growth medium was changed after 16 h later. 24 h and 48 h post transfection, cells were collected using Trypsin-EDTA (Gibco, #25200056), washed with PBS, and used for genomic DNA isolation.

tag: cell line

tag: cell type

value: ES cells

tag: construct

value: iPCR


title: miR-146a-/- ESLAM_CAACCCC

description:
C8AP5ANXX_1_1_CAACCCC_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: E14 cells, TG_LA_no_ISceI_rep4

description: UMI-amplicon

value: TG

tag: assay

value: LA

tag: time


title: miR-146a-/- ESLAM_AACCCAG

description:
C8AP5ANXX_1_1_AACCCAG_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: E14 cells, iPCR_3

description: inverse PCR


title: muscle_Smn-/-;SMN2_Pip6a-Scrambled_P7_rep3

type: RNA

city: Cardiff

first: Jimena

last: Monzón-Sandoval

address: Maindy Road

country: United Kingdom

institute: UK Dementia Research Institute at Cardiff University

description: GeneChip Mouse Transcriptome Assay 1.0 arrays were used (Affymetrix core facility, Karolinska Institute) with 100 ng of RNA per sample

hyb_protocol: Samples were hybridized using standard Affymetrix hybridization kit materials

scan_protocol: Arrays were scanned in an Affymetrix Scanner 3000

data_processing:
Annotations for the Mouse Transcript Array 1.0 at the transcript level were obtained from the Affymetrix website (http://www.affymetrix.com/products_services/arrays/specific/mo_trans_assay.  affx#1_4). Background correction and RMA normalization at the probe level using oligo R package. We summarized the data in ensemble transcript IDs using the average normalized data. For differential expression analysis, we used limma R package and considered a transcript differentially expressed if their FDR <0.05. A gene was considered differentially expressed if at least one of the associated transcripts was differentially expressed.
normalized data file includes:  RMA signal intensities summarized at the transcript level (Ensembl Transcript IDs)

label: biotin

molecule: total RNA

source_name: tibialis anterior of Smn-/-;SMN2 treated with Pip6a-Scrambled at P7

label_protocol: Biotin

extract_protocol: RNA was extracted by guanidinium thiocyantate-acid-phenol-chloroform extraction using TRIzol Reagent (Life Technologies) as per manufacturer’s instructions

treatment_protocol: Smn-/-;SMN2 mice received a facial intravenous injection at postnatal day (P) 0 and P2 of Pip6a-scrambled or Pip6a-PMO (10 μg/g)

value: tibialis anterior

tag: treatment

value: FVB/N


title: muscle_Smn-/-;SMN2_Pip6a-Scrambled_P7_rep1


title: muscle_Smn-/-;SMN2_Pip6a-Scrambled_P7_rep2


title: E14 cells, TG_LA_48h_rep3

description: UMI-amplicon


title: muscle_Smn-/-;SMN2_Pip6a-PMO_P7_rep2

source_name: tibialis anterior of Smn-/-;SMN2 treated with Pip6a-PMO at P7


title: muscle_Smn-/-;SMN2_Pip6a-PMO_P7_rep1


title: E14 cells, TG_LA_no_ISceI_rep1

description: UMI-amplicon


title: E14 cells, TG_LA_no_ISceI_rep2

description: UMI-amplicon


title: muscle_Smn-/-;SMN2_Pip6a-PMO_P7_rep3


title: E14 cells, TG_LA_no_ISceI_rep3

description: UMI-amplicon


title: E14 cells, TG_LA_48h_rep4

description: UMI-amplicon


title: muscle_WT_Untreated_P7_rep3

source_name: tibialis anterior of WT at P7

value: Wild-Type


title: muscle_WT_Untreated_P7_rep2


title: E14 cells, TG_LA_48h_rep1

description: UMI-amplicon


title: E14 cells, TG_LA_48h_rep2

description: UMI-amplicon


title: E14 cells, TG_LA_24h_rep4

description: UMI-amplicon


title: E14 cells, TG_LA_24h_rep3

description: UMI-amplicon


title: muscle_Smn-/-;SMN2_Untreated_P7_rep3

source_name: tibialis anterior of Smn-/-;SMN2 at P7


title: muscle_Smn-/-;SMN2_Untreated_P7_rep2


title: muscle_Smn-/-;SMN2_Untreated_P7_rep1


title: E14 cells, TG_LA_24h_rep2

description: UMI-amplicon


title: E14 cells, TG_6xPCR_no_ISceI_rep3

description: UMI-amplicon


title: muscle_WT_Untreated_P2_rep2

source_name: tibialis anterior of WT at P2


title: E14 cells, TG_6xPCR_no_ISceI_rep2

description: UMI-amplicon


title: E14 cells, TG_6xPCR_no_ISceI_rep4

description: UMI-amplicon


title: muscle_WT_Untreated_P7_rep1


title: muscle_Smn-/-;SMN2_Untreated_P2_rep3

source_name: tibialis anterior of Smn-/-;SMN2 at P2


title: E14 cells, TG_6xPCR_48h_rep2

description: UMI-amplicon


title: E14 cells, TG_6xPCR_no_ISceI_rep1

description: UMI-amplicon


title: muscle_Smn-/-;SMN2_Untreated_P2_rep2


title: E14 cells, TG_6xPCR_48h_rep4

description: UMI-amplicon


title: muscle_WT_Untreated_P2_rep3


title: E14 cells, TG_LA_24h_rep1

description: UMI-amplicon


title: E14 cells, TG_6xPCR_24h_rep3

description: UMI-amplicon


title: muscle_Smn-/-;SMN2_Untreated_P2_rep1


title: E14 cells, TG_6xPCR_48h_rep3

description: UMI-amplicon


title: E14 cells, TG_6xPCR_48h_rep1

description: UMI-amplicon


title: miR-146a-/- ESLAM_AACCGGC

description:
C8AP5ANXX_1_1_AACCGGC_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice


title: Melanotic Xp11 neoplasm-10

city: Nanjing

first: xiaotong

last: Wang

state: Jiangsu

address: Zhongshan East Road 305

department: Pathology

country: China

institute: Nanjing Jinlin Hospital

library_source: transcriptomic

data_processing:
The raw sequence was acquired by NGS. Low-quality reads were discarded by trimmomatic (version v0.36), aimed to trimming adaptors and eliminating poor-quality bases followed by rRNA and tRNA sequences removing.
The clean reads were mapped with STAR (version 2.5.3a) to the hg38 genome assembly (UCSC Genome Browser build). Transcripts and genes were quantified by RSEM33 (version v1.1.17).
We used FPKM (fragments per kilobase per million reads) to normalize gene abundance considering differences in sequencing depth and gene length.
Genome_build: hg38
Supplementary_files_format_and_content: Tab-delimited text files include FPKM values for each Sample.

organism: Homo sapiens

source_name: tumor

extract_protocol:
Total RNA was extracted from formalin-fixed, paraffin-embedded (FFPE)  samples after xylene deparaffinization using the RNeasy formalin-fixed paraffin-embedded kit (QIAGEN). Ribosomal RNA was depleted using RNase H followed by library preparation using the KAPA Stranded RNA-seq Kit with RiboErase (HMR) (KAPA Biosystems).
Library concentration was performed using a KAPA Library Quantification Kit (KAPA Biosystems), and the library quality was accessed using an Agilent High Sensitivity DNA kit and Bioanalyzer 2100 (Agilent Technologies), followed by sequencing on Illumina HiSeq next-generation sequencing (NGS) platforms (Illumina).

value: soft tissue

tag: sample type

value: FFPE sample


title: E14 cells, TG_6xPCR_24h_rep2

description: UMI-amplicon


title: Melanotic Xp11 neoplasm-14


title: Melanotic Xp11 neoplasm-11


title: Melanotic Xp11 neoplasm-12


title: muscle_WT_Untreated_P2_rep1


title: E14 cells, TG_6xPCR_24h_rep1

description: UMI-amplicon


title: Alveolar soft part sarcoma-8


title: Melanotic Xp11 neoplasm-9


title: E14 cells, TC_LA_no_ISceI_rep8

description: UMI-amplicon

value: TC


title: Normal tissue-7

source_name: normal tissue

value: kidney


title: E14 cells, TG_6xPCR_24h_rep4

description: UMI-amplicon


title: Normal tissue-6


title: E14 cells, TC_LA_no_ISceI_rep7

description: UMI-amplicon


title: Normal tissue-5


title: E14 cells, TC_LA_no_ISceI_rep6

description: UMI-amplicon


title: E14 cells, TC_LA_no_ISceI_rep5

description: UMI-amplicon


title: E14 cells, TC_LA_no_ISceI_rep4

description: UMI-amplicon


title: Normal tissue-4


title: E14 cells, TC_LA_no_ISceI_rep3

description: UMI-amplicon


title: Normal tissue-2


title: E14 cells, TC_LA_no_ISceI_rep2

description: UMI-amplicon


title: Normal tissue-1


title: E14 cells, TC_LA_no_ISceI_rep1

description: UMI-amplicon


title: E14 cells, TC_LA_48h_rep4

description: UMI-amplicon


title: Alveolar soft part sarcoma-6


title: Alveolar soft part sarcoma-7


title: Alveolar soft part sarcoma-3


title: Alveolar soft part sarcoma-4


title: E14 cells, TC_LA_48h_rep3

description: UMI-amplicon


title: E14 cells, TC_LA_48h_rep1

description: UMI-amplicon


title: E14 cells, TC_LA_24h_rep4

description: UMI-amplicon


title: E14 cells, TC_LA_24h_rep3

description: UMI-amplicon


title: Alveolar soft part sarcoma-5


title: E14 cells, TC_LA_24h_rep2

description: UMI-amplicon


title: Melanotic Xp11 neoplasm-8


title: Alveolar soft part sarcoma-2


title: E14 cells, TC_LA_48h_rep2

description: UMI-amplicon


title: Melanotic Xp11 neoplasm-7


title: E14 cells, TC_6xPCR_no_ISceI_rep8

description: UMI-amplicon


title: Melanotic Xp11 neoplasm-6


title: E14 cells, TC_LA_24h_rep1

description: UMI-amplicon


title: E14 cells, TC_6xPCR_no_ISceI_rep6

description: UMI-amplicon


title: Melanotic Xp11 neoplasm-3


title: Melanotic Xp11 neoplasm-5


title: E14 cells, TC_6xPCR_no_ISceI_rep5

description: UMI-amplicon


title: PEComa-8


title: PEComa-9

value: bladder


title: E14 cells, TC_6xPCR_no_ISceI_rep2

description: UMI-amplicon


title: Alveolar soft part sarcoma-1


title: E14 cells, TC_6xPCR_no_ISceI_rep3

description: UMI-amplicon


title: PEComa-5


title: PEComa-6


title: E14 cells, TC_6xPCR_no_ISceI_rep1

description: UMI-amplicon


title: E14 cells, TC_6xPCR_48h_rep4

description: UMI-amplicon


title: PEComa-4

value: intestine


title: E14 cells, TC_6xPCR_48h_rep3

description: UMI-amplicon


title: E14 cells, TC_6xPCR_24h_rep4

description: UMI-amplicon


title: E14 cells, TC_6xPCR_48h_rep1

description: UMI-amplicon


title: Normal tissue-3


title: E14 cells, TC_6xPCR_48h_rep2

description: UMI-amplicon


title: PEComa-2

value: lung


title: E14 cells, TC_6xPCR_24h_rep3

description: UMI-amplicon


title: PEComa-3


title: PEComa-1


title: Xp11 renal cell carcinoma-12


title: PEComa-7

value: uterus


title: E14 cells, TC_6xPCR_no_ISceI_rep7

description: UMI-amplicon


title: E14 cells, TC_6xPCR_24h_rep2

description: UMI-amplicon


title: Melanotic Xp11 neoplasm-2


title: Xp11 renal cell carcinoma-11


title: E14 cells, CA_LA_no_ISceI_rep4

description: UMI-amplicon

value: CA


title: MITF renal cell carcinoma


title: Xp11 renal cell carcinoma-9


title: Xp11 renal cell carcinoma-10


title: Xp11 renal cell carcinoma-8


title: E14 cells, CA_LA_no_ISceI_rep3

description: UMI-amplicon


title: E14 cells, CA_LA_no_ISceI_rep1

description: UMI-amplicon


title: E14 cells, CA_LA_no_ISceI_rep2

description: UMI-amplicon


title: E14 cells, CA_LA_48h_rep3

description: UMI-amplicon


title: E14 cells, CA_LA_48h_rep4

description: UMI-amplicon


title: Xp11 renal cell carcinoma-7


title: E14 cells, CA_LA_48h_rep1

description: UMI-amplicon


title: Xp11 renal cell carcinoma-2


title: Xp11 renal cell carcinoma-3


title: E14 cells, CA_LA_24h_rep4

description: UMI-amplicon


title: Xp11 renal cell carcinoma-6


title: Xp11 renal cell carcinoma-5


title: Xp11 renal cell carcinoma-1


title: E14 cells, TC_6xPCR_24h_rep1

description: UMI-amplicon


title: E14 cells, CA_LA_24h_rep3

description: UMI-amplicon


title: E14 cells, CA_6xPCR_no_ISceI_rep4

description: UMI-amplicon


title: E14 cells, CA_LA_24h_rep1

description: UMI-amplicon


title: Melanotic Xp11 neoplasm-1


title: E2+TH+Tam_3

city: Cleveland

first: Hyunmin

last: Kim

state: Ohio

address: 10900 Euclid Ave.

department: Department of Genetics and Genome Sciences

institute: Case Western Reserve University

description:
sample name:
28L

data_processing:
Hisat2 (version 2.1.0) ssed to map reads to human genome GRCh37
bedtools used to assemble transcripts
edgeR used to identify differenentially expressed genes
Genome_build: GRCH37/hg19
Supplementary_files_format_and_content: tab-delimited text file includes raw and normalized expression

source_name: Breast tumor xenograft

growth_protocol: Tumors were excised from mice and snap frozen in liquid nitrogen

extract_protocol:
RNA was extraced from pulverized tissue using Trizol followed by RNeasy kit (Qiagen) column cleanup and DNAase digestion
RNAlibraries were prepared for sequencing using TruSEQ RNA Sample Prep v4 using standard Illumina protocols.

treatment_protocol: Tumors were excised from mice treated with thyroid hormone or tamoxifen with and without estradiol supplementation

tag: xenograft

value: estrogen + thyroid hormone+tamoxifen

value: breast tumor


title: E2+TH+Tam_5

description:
sample name:
38L


title: E2+TH+Tam_2

description:
sample name:
14R


title: E14 cells, CA_6xPCR_no_ISceI_rep3

description: UMI-amplicon

value: ES cells


title: E2+TH+Tam_6

description:
sample name:
39L


title: E14 cells, CA_6xPCR_48h_rep3

description: UMI-amplicon


title: E2+Tam_6

description:
sample name:
25L

value: estrogen + tamoxifen


title: E14 cells, CA_6xPCR_48h_rep2

description: UMI-amplicon


title: E14 cells, CA_LA_48h_rep2

description: UMI-amplicon

value: LA


title: E2+Tam_5

description:
sample name:
24L


title: E2+Tam_4

description:
sample name:
22L


title: E14 cells, CA_6xPCR_no_ISceI_rep1

description: UMI-amplicon


title: E14 cells, CA_6xPCR_no_ISceI_rep2

description: UMI-amplicon


title: E2+TH+Tam_4

description:
sample name:
36L


title: Xp11 renal cell carcinoma-4


title: E14 cells, CA_6xPCR_24h_rep4

description: UMI-amplicon


title: E2+Tam_3

description:
sample name:
21L


title: E14 cells, CA_6xPCR_48h_rep4

description: UMI-amplicon


title: miR-146a-/- ESLAM_AAGCCCA

description:
C8AP5ANXX_1_1_AAGCCCA_125bp.concat_chastity_passed
ESLAM cells isolated from pooled 10-14 week-old mice

tag: strain background

value: 10-14 weeks

value: bone marrow

tag: cells

value: ESLAM (EPCR+ CD45+ CD150+ CD48-)

value: Post-Bisulfite Adapter Ligation DNA


title: E14 cells, CA_LA_24h_rep2

description: UMI-amplicon


title: E2+Tam_2

description:
sample name:
19L


title: E14 cells, CA_6xPCR_48h_rep1

description: UMI-amplicon


title: E2+TH+Tam_1

description:
sample name:
12R


title: E14 cells, CA_6xPCR_24h_rep3

description: UMI-amplicon


title: E2+TH_6

description:
sample name:
4R

value: estrogen + thyroid hormone


title: E14 cells, CA_6xPCR_24h_rep2

description: UMI-amplicon


title: E2+TH_3

description:
sample name:
26R


title: E14 cells, CA_6xPCR_24h_rep1

description: UMI-amplicon


title: E14 cells, AG_LA_no_ISceI_rep7

description: UMI-amplicon

value: AG


title: E14 cells, AG_LA_no_ISceI_rep8

description: UMI-amplicon


title: E14 cells, AG_LA_no_ISceI_rep6

description: UMI-amplicon


title: E14 cells, AG_LA_no_ISceI_rep4

description: UMI-amplicon


title: E2+TH_2

description:
sample name:
1R


title: E2+TH_1

description:
sample name:
1L


title: E14 cells, AG_LA_no_ISceI_rep3

description: UMI-amplicon


title: E14 cells, AG_LA_no_ISceI_rep2

description: UMI-amplicon


title: E2_6

description:
sample name:
7R

value: estrogen


title: E14 cells, AG_LA_48h_rep4

description: UMI-amplicon


title: E14 cells, AG_LA_48h_rep3

description: UMI-amplicon


title: E2_5

description:
sample name:
6R


title: E2_3

description:
sample name:
20R


title: E14 cells, AG_LA_no_ISceI_rep1

description: UMI-amplicon


title: E14 cells, AG_LA_48h_rep2

description: UMI-amplicon


title: E2_4

description:
sample name:
31L


title: E2_2

description:
sample name:
17R


title: E14 cells, AG_LA_48h_rep1

description: UMI-amplicon


title: E2_1

description:
sample name:
10R


title: E14 cells, AG_LA_no_ISceI_rep5

description: UMI-amplicon


title: E14 cells, AG_LA_24h_rep4

description: UMI-amplicon


title: E14 cells, AG_LA_24h_rep3

description: UMI-amplicon


title: WT_untreated_rep3

first: Viola

last: Volpato

address: Maindy Rd

department: UK DRI

institute: Cardiff University

data_processing:
Quality control of the sequencing: FastQC v0.11.7
Indexing of the genome (transcripts): kallisto index v0.44.0; kmer-size=31
Pseudoalignment with kallisto using the reads: kallisto quant v0.44.0;
Genome_build: mm10
Supplementary_files_format_and_content: Matrix table with gene counts for every gene and every sample from the pseudoalignment using Kallisto quant

molecule: polyA RNA

source_name: Skeletal muscle

extract_protocol:
RNA was extracted using a RNeasy Microarray Tissue Mini Kit from Qiagen. Lysis and homogenization were performed using QIAzol Lysis Reagent
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Harmine hydrochloride (sc-295136, Insight Biotechnology Ltd, Sante Cruz) was dissolved in 0.9% saline and administered daily (10 mg/kg) by gavage.

value: Skeletal muscle

value: FVB/N

value: post natal day 7

tag: genotype

value: WT

value: Untreated


title: Melanotic Xp11 neoplasm-4

value: kidney

value: FFPE sample


title: WT_treated_rep3

tag: age

value: Harmine treated


title: E14 cells, AG_LA_24h_rep1

description: UMI-amplicon

value: ES cells


title: E14 cells, TC_6xPCR_no_ISceI_rep4

description: UMI-amplicon

value: TC


title: WT_treated_rep1

tag: tissue


title: E14 cells, AG_6xPCR_no_ISceI_rep7

description: UMI-amplicon


title: WT_treated_rep2


title: E14 cells, AG_6xPCR_no_ISceI_rep5

description: UMI-amplicon


title: E14 cells, AG_6xPCR_no_ISceI_rep6

description: UMI-amplicon


title: E14 cells, AG_6xPCR_no_ISceI_rep4

description: UMI-amplicon


title: E14 cells, AG_LA_24h_rep2

description: UMI-amplicon

value: LA


title: E14 cells, AG_6xPCR_no_ISceI_rep3

description: UMI-amplicon


title: SMA_treated_rep3


title: SMA_untreated_rep3


title: E14 cells, AG_6xPCR_no_ISceI_rep2

description: UMI-amplicon


title: Melanotic Xp11 neoplasm-13

value: soft tissue


title: SMA_treated_rep2


title: SMA_treated_rep1


title: WT_untreated_rep2


title: SMA_untreated_rep1


title: WT_untreated_rep1


title: E14 cells, AG_6xPCR_48h_rep2

description: UMI-amplicon


title: E14 cells, AG_6xPCR_24h_rep4

description: UMI-amplicon


title: E14 cells, AG_6xPCR_48h_rep1

description: UMI-amplicon


title: SMA_untreated_rep2


title: WT LSK [C6PVKANXX_7_ATCACG]

description:
C6PVKANXX_7_1_ATCACG
LSK cells isolated from pool of 12 10-14 week-old mice

data_processing:
Alignment using STAR (v2.5.3a)
Expression called using salmon (v0.9.0)
Genome_build: mm10
Supplementary_files_format_and_content: counts

source_name: mouse bone marrow

extract_protocol:
AllPrep DNA/RNA Mini Kit (Qiagen, 80204)
single-stranded, ribodepleted

value: 10-14 weeks

value: bone marrow

value: Lin- Sca-1+ c-Kit+ cells


title: E14 cells, AG_6xPCR_24h_rep3

description: UMI-amplicon

value: AG


title: E14 cells, AG_6xPCR_no_ISceI_rep1

description: UMI-amplicon


title: miR-146a-/- LSK [C6PVKANXX_7_AACCCC]

description:
C6PVKANXX_7_1_AACCCC
LSK cells isolated from pool of 6 10-14 week-old mice


title: miR-146a-/- LSK WGBS

description:
C5DMUANXX_*_AAGACT
C5L25ANXX_*_AAGACT
LSK cells isolated from pool of 6 10-14 week-old mice

data_processing:
The first 6 bases of read1 and read2 were trimmed using Trimgalore v0.4.0 and Cutadapt v1.2.143 using the parameters --clip_R1 6 --clip_R2 6 -paired. Trimmed fastq files were aligned using Novoalign version V3.02.10 (www.novocraft.com) to the mouse assembly GRCm38 (mm10). Alignments were done in paired-end mode using the options -b4 (non-directional), -t 20,3 (optimized for SNP concordance), -a (adapter trimming), --hlimit 8 (homopolymer filter), and -H 20 (removes trailing bases with quality ² 20), and -u8 (penalty for unconverted CHG or CHH cytosine) as recommended by Novoalign. Aligned reads were output into the BAM format and sorted using SAMtools V0.1.17.
Duplicates were marked using Picard (1.31) and removed. CpG methylation status was called using Samtools (0.1.13) mpileup and Novomethyl (1.01), according to the protocol specified by Novocraft (http://www.novocraft.com/documentation/novoalign-2/novoalign-user-guide/bisulphite-treated-reads/novomethyl-analyzing-methylation-status/). CHG and CHH calls were removed, as well as all CpG calls with <5 reads. The remaining CpG calls were converted into separate bigWIG files, containing per-CpG methylation and coverage, respectively.
Genome_build: mm10
Supplementary_files_format_and_content: bigwig

extract_protocol:
Bone marrow was harvested from the femur, tibia and pelvic bones of 10-14 week old, mixed sex C57BL/6 mice, pooled, and subjected to ammonium chloride mediated red blood cell lysis. The sample was stained with a Lineage/Sca1/Kit staining cocktail, and 10,000 Lin-Sca1+Kit- cells were FACS sorted into an Eppendorf tube, and DNA was extracted using AllPrep DNA/RNA Mini Kit (Qiagen, 80204) according to manufacturer’s instructions.
4 uL of purified DNA (25 ng/uL) was mixed with 1 uL of T7 Phage Lambda DNA spikeins (1 ng/uL), and the mixture was used directly as input to bisulfite conversion and desulphonation as described for single-cells, except neither DNase nor UV decontamination was performed on the reagents, and 5 uL of beads was used instead of 1.8 uL. After bisulfite conversion and desulphonation, the DNA was eluted in 40 uL of EB buffer before used as input for one round of double-stranding with 2.5 uL of 10 mM dNTPs, 2 uL of 500 uM random hexamers, 1 uL of 50 U/uL Klenow exo- (NEB, M0212M) and 5 uL of 10X NEB Buffer 2. Library construction was the same for single-cells except only 4 rounds of PCR was used. Libraries were ran on agilent and sequenced directly without qPCR quality control.

value: Lin-Sca1+Kit- (LSK) cells

tag: molecule subtype

value: Bisulfite-treated DNA


title: E14 cells, AC_LA_no_ISceI_rep4

description: UMI-amplicon

value: AC


title: E14 cells, AG_6xPCR_24h_rep1

description: UMI-amplicon


title: Gln_4

city: Duarte

first: Haiqing

last: Li

state: CA

address: 1500 East Duarte Road

department: Department of Computational & Quantitative Medicine

institute: Beckman Research Institute of City of Hope, Center for Informatics

description: melanoma tumor tissue

data_processing:
Basecalls performed using CASAVA version 1.8
The RNA-seq sequence reads were mapped to mouse genome assembly hg19 using open source RNA-seq alignment tool HISAT2 (ver 2.0.4)
The alignment results were converted to RNA-seq gene expression measurement as RPKM (reads/kilo base of total exon length/million mapped) using Partek Genome Suite (version 6.6) with NCBI RefSeq database.
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for all Samples

source_name: Melanoma

growth_protocol: PDX tumors were subcutanously implanted in nude mice. Tumors were collected for RNA extraction and sequencing when tumor reached 1 cm3

extract_protocol:
Total RNA was extracted using Trizol reagent (15596026, Life Technologies, Carlsbad, CA, USA) from tumour tissues.
RNA sequencing libraries were prepared with Kapa RNA mRNA HyperPrep kit (Kapa Biosystems, Cat KR1352) according to the manufacturer's protocol.

treatment_protocol: Mice were placed on control or high glutamine diet one week post tumor implantation.

tag: tumor model

value: Melanoma PDX TM00702 model (Jackson Lab)

value: High glutamine diet

value: Melanoma


title: E14 cells, AC_LA_no_ISceI_rep3

description: UMI-amplicon


title: Gln_3

description: melanoma tumor tissue


title: E14 cells, AC_LA_no_ISceI_rep2

description: UMI-amplicon


title: E14 cells, AG_6xPCR_24h_rep2

description: UMI-amplicon


title: Gln_2

description: melanoma tumor tissue


title: E14 cells, AC_LA_no_ISceI_rep1

description: UMI-amplicon


title: Gln_1

description: melanoma tumor tissue


title: E14 cells, AC_LA_48h_rep4

description: UMI-amplicon


title: Ctrl_4

description: melanoma tumor tissue

value: Control diet


title: Ctrl_3

description: melanoma tumor tissue


title: SDC1-silenced group_caco2_rep1

city: Singapore

first: George

middle: W

last: Yip

address: 4 Medical Drive, Block MD10

country: Singapore

institute: National University of Singapore

description: Gene expression data from Caco-2 cells transfected with SDC1 siRNA

hyb_protocol: Fragmented cRNA was hybridised at 45°C for 16 hours to Affymetrix HG U133 Plus 2.0 GeneChips, followed by washing and staining in an Affymetrix FS450 fluidics station.

scan_protocol: GeneChips were scanned using a GCOS 7000 7G scanner.

data_processing: Data were analysed using GeneSpring GX11.0 with RMA normalisation to median signal intensity.

label: Biotin

label_protocol: Biotinylated cRNA was prepared according to the standard Affymetrix protocol from 3.6 µg of total RNA.

growth_protocol: Caco-2 cells were cultured at 37°C in a humidified atmosphere with 5% carbon dioxide.

extract_protocol: Total RNA extracted using the Qiagen RNeasy Kit.

treatment_protocol: Cells were transfected with SDC1-siRNA or a negative control siRNA.

value: Human colorectal adenocarcinoma


title: NbBsrDI-nickase-TdT

first: Xiaolin

last: Wu

state: Singapore

address: 1 CREATE Way

institute: Singapore-MIT Alliance for Research and Technology (SMART)

data_processing:
Sequencing results were processed on the Galaxy web platform (https://usegalaxy.org/). Initially, the paired end reads were pre-processed by trim galore to remove adapters and the first 3 bp on the 5’ of read 1 were also trimmed.
All the reads were aligned to the corresponding genome using Bowtie 2.
A custom method for peak calling of sequencing data was developed with BamTools, BEDTools and Rstudio. Briefly, the bam results were filtered based on R1 (selected for nick translation data) or R2 (selected for 3’ capture data). The 5’ coverage (experiment sample) or full coverage (controls) on each position were calculated based on the filtered bam result by BEDTools(positive and negative strand separately)
The coverage of positionN (sample)/ coverage of positionN-1(sample), coverage of positionN(sample)/ coverage of positionN+1(sample) and coverage of positionN(sample)/ coverage of positionN(control)  ratio was calculated on each position and the positions with all the three ratios above 5 were regarded as strong signals.
Genome_build: NC_010473.1
Supplementary_files_format_and_content: tabular files were generated by BEDTools containing the 5’ coverage (experiment sample) or full coverage (controls) on each position (positive and negative strand separately)

organism: Escherichia coli str. K-12 substr. DH10B

source_name: bacteria

growth_protocol: E. coli K12 strains were grown in Luria-Bertani (LB) at 37 °C.

extract_protocol:
R1:Nick translation of genomic DNA. After creation or conversion of the interested sites into nick sites, split the DNA samples equally into two tubes: one for nick translation and the other for negative control. The nick translation was performed in a 50 μl reaction system containing 2 μl of alpha-thio-dNTPs (2.5 mM each, TRILINK), 1X of NEBcutsmart buffer, 2 μl of DNA polymerase I and DNA template. For the negative control, used H2O instead of DNA polymerase I. The reaction mixture was incubated at 15 °C for 90 min and then terminated by heating at 75 °C for 20 min. The product was ready of template DNA digestion after the purification by DyeEx.  Genomic DNA template digestion. The DNA product from nick translation was digested by Exonuclease III (NEB) and RecJf (NEB) to remove the template DNA. A 50 μl reaction system containing 200 units Exonuclease III, 5 μl NEBcutsmart buffer and DNA product from nick translation was incubated at 37 °C for 60 min to digest double-stranded DNA template. Incubate the reaction at 95 °C for 3 min and then put on ice immediately to denature DNA. And then add 60 units of RecJf, incubate at 37 °C for 60 min. For some DNA samples with high complexity of structure and/or modifications, additional digestion with 60 units of RecJf might be necessary. After digestion, inactivate the enzymes by incubation at 80 °C for 10 min. The product was then purified by Zymo Oligo clean & concentrator kit (Zymo) following the manufacturer’s protocol. The purified product was ready for Illumina library preparation.   Nick translation of genomic DNA. After creation or conversion of the interested sites into nick sites, split the DNA samples equally into two tubes: one for nick translation and the other for negative control. The nick translation was performed in a 50 μl reaction system containing 2 μl of alpha-thio-dNTPs (2.5 mM each, TRILINK), 1X of NEBcutsmart buffer, 2 μl of DNA polymerase I and DNA template. For the negative control, used H2O instead of DNA polymerase I. The reaction mixture was incubated at 15 °C for 90 min and then terminated by heating at 75 °C for 20 min. The product was ready of template DNA digestion after the purification by DyeEx.  Genomic DNA template digestion. The DNA product from nick translation was digested by Exonuclease III (NEB) and RecJf (NEB) to remove the template DNA. A 50 μl reaction system containing 200 units Exonuclease III, 5 μl NEBcutsmart buffer and DNA product from nick translation was incubated at 37 °C for 60 min to digest double-stranded DNA template. Incubate the reaction at 95 °C for 3 min and then put on ice immediately to denature DNA. And then add 60 units of RecJf, incubate at 37 °C for 60 min. For some DNA samples with high complexity of structure and/or modifications, additional digestion with 60 units of RecJf might be necessary. After digestion, inactivate the enzymes by incubation at 80 °C for 10 min. The product was then purified by Zymo Oligo clean & concentrator kit (Zymo) following the manufacturer’s protocol. The purified product was ready for Illumina library preparation.   R2:Mapping of modification/damage sites on DNA by TdT-dependent method. The steps of DNA fragmentation, blocking and nick conversion were same as described above. 100ng of the nick converted DNA was denatured by heating at 95 °C for 3 min in 20 μl H2O, followed by Illumina library preparation with the Clontech SMART ChIp-seq kit (Clontech) by following the manufacturer’s protocol. 12 cycles were used in the final step of PCR amplification.
Illumina library preparation. Illumina library preparation was performed by the Clontech SMART ChIp-seq kit (Clontech) by following the manufacturer’s protocol. 12 cycles were used in the final step of PCR amplification. The PCR product from each sample was combined with its corresponding negative control and then size selected using AMPure XP beads (NEB). The purified library was submitted to Illumina instrument for paired-end sequencing.

treatment_protocol: 1.Fragmentation of genomic DNA.  To ensure a relatively random fragmentation of genomic DNA, 1 μg of the extracted DNA was digested separately by three DNA endonuclease combinations: NciI; HindIII and XhoI; SalI, XbaI and NdeI. RNase A was also added to each digestion mixture to remove any trace of contaminated RNA. After digestion, the digested products were purified by Qiagen PCR purification Kit. The three purified DNA sample were mixed together for the following blocking step.  2.Blocking of pre-existing strand break sites. Reaction mixture (40 μl) containing 4 μl of reaction buffer (NEBcutsmart buffer); 1 μl of Shrimp Alkaline Phosphatase (rSAP, NEB), 1 μg of template genomic DNA was incubated at 37 °C for 30 min to remove any pre-existing strand breaks with 3' end phosphate, then 70°C for 10min to inactivate rSAP. After cool down the reaction product to room temperature, add 2 μl of ddNTPs (2.5 mM each, TriLink) and 1 μl of DNA polymerase I (10 U, NEB), incubate at 37 °C for 40 min to block any pre-existing strand break sites. Then 1 μl of Shrimp Alkaline Phosphatase (rSAP, NEB) was added to the reaction mixture, incubate at 37 °C for another 30 min to degrade the excess ddNTPs. Reaction was terminated by heat-inactivation of rSAP and DNA polymerase I at 75 °C for 10 min. The reaction product was ready for one of the following nick creation or conversion procedure after de-salts by DyeEx column (QIAGEN)  3.Nick creation of E. coli DH10B genomic DNA with Nb. BsmI and Nb. BsrDI. A 50 μl reaction mixture containing 1 μl Nb. BsmI (10 U, NEB), 1 μl Nb. BsrDI (10 U, NEB), 1 μg genomic DNA and 1X NEBcutsmart buffer was incubated at 65 °C for 1 hour. The reaction was terminated by heating at 80 °C for 20 minutes and cooled down to 4 °C at the rate of 0.1 °C/s. The reaction product was used for nick translation as described below with no further purification.

tag: role

value: experiment sample

tag: library prep

value: R2-TdT dependent


title: NbBsrDI-NO-TdT

value: negative control


title: NbBsrDI-nickase-NT

value: R1-Nick translation


title: SDC1-silenced group_caco2_rep3

source_name: Caco-2 cells transfected with SDC1 siRNA

tag: cell line


title: Control group_caco2_rep3

description: Gene expression data from Caco-2 cells transfected with negative control siRNA

tag: cell type


title: SDC1-silenced group_caco2_rep2


title: E14 cells, AC_LA_48h_rep3

description: UMI-amplicon

value: ES cells

tag: construct

value: LA


title: E2+Tam_1

description:
sample name:
18R

value: estrogen + tamoxifen

value: breast tumor


title: Control group_caco2_rep1

source_name: Caco-2 cells transfected with negative control siRNA


title: Control group_caco2_rep2


title: Ctrl_2

description: melanoma tumor tissue


title: 0.2mM-NO-TdT

growth_protocol: A single colony of E. coli DH10B was grown in 5 mL LB medium overnight at 37 °C. 1mL cells were pelleted were harvested by centrifuge at ambient temperature (unless indicated otherwise). Cells were resuspended and diluted with fresh 10 LB medium to a starting optical density at 600 nm (OD600) of 0.1, followed by growth at 37°C, 230 rpm until OD600=0.8. 10 μL diluted H2O2 solution were added to the culture with a final concentration 0.1, 0.5, 1 and 2 mM. As un-exposed control, 10 μL sterile water was used instead of H2O2. After sitting at ambient temperature for 30 min, 10 μL of the cells were used for lethal dose (LD) analysis by counting the colony formation unit on LB agar plate. All the rest cells were harvested for DNA extraction with OMEGA bacterial genomic DNA isolation kit by following the manufacture’s protocol.

treatment_protocol: 1.Fragmentation of genomic DNA.  To ensure a relatively random fragmentation of genomic DNA, 1 μg of the extracted DNA was digested separately by three DNA endonuclease combinations: NciI; HindIII and XhoI; SalI, XbaI and NdeI. RNase A was also added to each digestion mixture to remove any trace of contaminated RNA. After digestion, the digested products were purified by Qiagen PCR purification Kit. The three purified DNA sample were mixed together for the following blocking step.  2.Blocking of pre-existing strand break sites. Reaction mixture (40 μl) containing 4 μl of reaction buffer (NEBcutsmart buffer); 1 μl of Shrimp Alkaline Phosphatase (rSAP, NEB), 1 μg of template genomic DNA was incubated at 37 °C for 30 min to remove any pre-existing strand breaks with 3' end phosphate, then 70°C for 10min to inactivate rSAP. After cool down the reaction product to room temperature, add 2 μl of ddNTPs (2.5 mM each, TriLink) and 1 μl of DNA polymerase I (10 U, NEB), incubate at 37 °C for 40 min to block any pre-existing strand break sites. Then 1 μl of Shrimp Alkaline Phosphatase (rSAP, NEB) was added to the reaction mixture, incubate at 37 °C for another 30 min to degrade the excess ddNTPs. Reaction was terminated by heat-inactivation of rSAP and DNA polymerase I at 75 °C for 10 min. The reaction product was ready for one of the following nick creation or conversion procedure after de-salts by DyeEx column (QIAGEN)  3.Nick conversion of DNA damage sites. In this study, we mapped the damage sites of genomic DNA extracted from H2O2-treated E. coli cells, including AP sites that can be recognized. To convert the interested damage sites into strand breaks, a 50 μl reaction mixture containing 1 μg genomic DNA, Endonuclease IV (20 U) or Fpg (16 U) enzymes, 1X NEBcutsmart buffer was incubated at 37 °C for 60 minutes. The reaction mixture was then kept on ice and used for the following nick translation procedure with no further inactivation or purification.


title: 0.2mM-EndoIV-TdT


title: E14 cells, AC_LA_48h_rep2

description: UMI-amplicon

tag: assay


title: Ctrl_1

description: melanoma tumor tissue


title: 0mM-EndoIV-TdT


title: E14 cells, AC_LA_48h_rep1

description: UMI-amplicon

tag: time


title: E14 cells, AC_LA_24h_rep4

description: UMI-amplicon


title: 0.2mM-EndoIV-NT

tag: treatment


title: 0mM-NO-TdT


title: E14 cells, AC_LA_24h_rep3

description: UMI-amplicon


title: mLN BEC

city: Hannover

first: Oliver

last: Dittrich-Breiholz

address: Carl-Neuberg-Str. 1

department: Research Core Unit Genomics

country: Germany

institute: Medical School Hannover

hyb_protocol: cRNA fragmentation, hybridisation and washing steps were carried-out as recommended in the ‘One-Color Microarray-Based Gene Expression Analysis Protocol V5.7’, except that 400ng of each fluorescently labelled cRNA population was used for hybridization.

scan_protocol: Slides were scanned using the Agilent Micro Array Scanner G2565CA (pixel resolution 5 µm, bit depth 20).

data_processing: Data extraction and normalization were performed with the “Feature Extraction Software V10.7.3.1” by using the recommended default extraction protocol file: GE1_107_Sep09.xml. Measurements of on-chip replicates were averaged using the geometric mean of processed intensity values of the green channel, ‘gProcessedSignal’ (gPS) to retrieve one resulting value per unique non-control probe. Single Features were excluded from averaging, if they i) were manually flagged, ii) were identified as Outliers by the Feature Extraction Software, iii) lay outside the interval of ‘1.42 x interquartile range‘ regarding the normalized gPS distribution of the respective on-chip replicate population, or, iv) showed a coefficient of variation of pixel intensities per Feature that exceeded 0.5.   Averaged gPS values were normalized by Quantile normalization aproach.  Finally, a lower intensity threshold (surrogate value) was defined based on intensity distribution of negative control features. This value was fixed at 15 normalized gPS units. All of those measurements that fell below this intensity cutoff were substituted by the respective surrogate value of 15.

source_name: blood endothelial cells isolated from mesenteric lymph node (gp38- CD31+)

label_protocol: 30-50ng of total RNA were used to prepare aminoallyl-UTP-modified (aaUTP) cRNA (Amino Allyl MessageAmp™ II Kit; #AM1753; Thermo Fisher Scientific) applying one-round of amplification as directed by the company, except for a twofold downscaling of all reaction volumes. The labeling of aaUTP-cRNA was performed by use of Alexa Fluor 555 Reactive Dye (#A32756; Thermo Fisher Scientific) as recommended in the manual of the Amino Allyl MessageAmp™ II Kit (twofold downscaled reaction volumes).

extract_protocol: Sorted cells from mLN were isolated and total RNA was isolated according to the manufacturer´s protocol (RNeasy Micro Kit, Qiagen, Hilden, Germany).

treatment_protocol: For isolation of CD45- stem cells, the mesenteric lymph node (mLN) of wildtyp mice were removed, and LNs were digested at 37° for 30 min with 1 mg/ml Collagenase 8 in RPMI 1640/10% FCS. CD45- cells were isolated using the MACS technique following the instructions provided by Miltenyi. Stromal cell subsets were sorted using fluorochrome-coupled antibodies (CD45- CD24- CD31 gp38). The mean purity of stromal cell subsets was 88% ± 1.5. The SCs were used for mRNA isolation.

tag: strain


title: E14 cells, AC_LA_24h_rep2

description: UMI-amplicon

source_name: ES cells


title: E14 cells, AC_LA_24h_rep1

description: UMI-amplicon


title: mLN LEC

source_name: lymphatic endothelial cells isolated from mesenteric lymph node (gp38+ CD31+)


title: E2+TH_4

description:
sample name:
27L

tag: xenograft

value: estrogen + thyroid hormone


title: E2+TH_5

description:
sample name:
4L


title: 0.2mM-NO-NT


title: 0mM-NO-NT


title: E14 cells, AC_6xPCR_no_ISceI_rep8

description: UMI-amplicon


title: mLN_exp2

scan_protocol: Slides were scanned on the Agilent Micro Array Scanner G2505 B at two different PMT settings (100 % and 5 %) to increase the dynamic range of the measurements (extended dynamic range mode).

data_processing: Data extraction and normalization were performed with the “Feature Extraction Software V9.5.3.1” by using the recommended default extraction protocol file: GE1-v5_95_Feb07.xml. Measurements of on-chip replicates were averaged using the geometric mean of processed intensity values of the green channel, ‘gProcessedSignal’ (gPS) to retrieve one resulting value per unique non-control probe. Single Features were excluded from averaging, if they i) were manually flagged, ii) were identified as Outliers by the Feature Extraction Software, iii) lay outside the interval of ‘1.42 x interquartile range‘ regarding the normalized gPS distribution of the respective on-chip replicate population, or, iv) showed a coefficient of variation of pixel intensities per Feature that exceeded 0.5.   Averaged gPS values were normalized by global linear scaling: All gPS values of one sample were multiplied by an array-specific scaling factor. This factor was calculated by dividing a ‘reference 75th Percentile value’ (set as 1500 for the whole series) by the 75th Percentile value of the particular Microarray to be normalized (‘Array I’ in the formula shown below). Accordingly, normalized gPS values for all samples (microarray data sets) were calculated by the following formula:   normalized gPSArray i = gPSArray i  x (1500 / 75th PercentileArray i)    Finally, a lower intensity threshold (surrogate value) was defined based on intensity distribution of negative control features. This value was fixed at 15 normalized gPS units. All of those measurements that fell below this intensity cutoff were substituted by the respective surrogate value of 15.

label_protocol: 80ng of total RNA were used to prepare aminoallyl-UTP-modified (aaUTP) cRNA (Amino Allyl MessageAmp™ II Kit; #AM1753; Thermo Fisher Scientific) applying one-round of amplification as directed by the company, except for a twofold downscaling of all reaction volumes. The labeling of aaUTP-cRNA was performed by use of Alexa Fluor 555 Reactive Dye (#A32756; Thermo Fisher Scientific) as recommended in the manual of the Amino Allyl MessageAmp™ II Kit (twofold downscaled reaction volumes).

extract_protocol: Sorted cells from pLN and mLN were isolated and total RNA was isolated according to the manufacturer´s protocol (RNeasy Micro Kit, Qiagen, Hilden, Germany).

treatment_protocol: For isolation of CD45- SCs, the mesenteric lymph node (mLN) or the peripheral lymph node (pLN) of wildtyp mice were removed, and LNs were digested at 37° for 30 min with 1 mg/ml Collagenase 8 in RPMI 1640/10% FCS. CD45- cells were isolated using the MACS technique following the instructions provided by Miltenyi. The mean purity of CD45- cells was 97.4% ± 2.0 for mLN cells and 97.5% ± 2.2 for pLN cells.


title: S87-NT-iodine

description: Salmonella enterica serovar Cerro 87

data_processing:
Sequencing results were processed on the Galaxy web platform (https://usegalaxy.org/). Initially, the paired end reads were pre-processed by trim galore to remove adapters and the first 3 bp on the 5’ of read 1 were also trimmed.
All the reads were aligned to the corresponding genome using Bowtie 2.
A custom method for peak calling of sequencing data was developed with BamTools, BEDTools and Rstudio. Briefly, the bam results were filtered based on R1 (selected for nick translation data) or R2 (selected for 3’ capture data). The 5’ coverage (experiment sample) or full coverage (controls) on each position were calculated based on the filtered bam result by BEDTools(positive and negative strand separately)
The coverage of positionN (sample)/ coverage of positionN-1(sample), coverage of positionN(sample)/ coverage of positionN+1(sample) and coverage of positionN(sample)/ coverage of positionN(control)  ratio was calculated on each position and the positions with all the three ratios above 5 were regarded as strong signals.
Genome_build: CP008925.1
Supplementary_files_format_and_content: tabular files were generated by BEDTools containing the 5’ coverage (experiment sample) or full coverage (controls) on each position (positive and negative strand separately)

organism: Salmonella enterica subsp. enterica serovar Cerro

growth_protocol: Salmonella enterica serovar Cerro 87strains were grown in Luria-Bertani (LB) at 37 °C.  All the rest cells were harvested for DNA extraction with OMEGA bacterial genomic DNA isolation kit by following the manufacture’s protocol.

treatment_protocol: 1.Fragmentation of genomic DNA.  To ensure a relatively random fragmentation of genomic DNA, 1 μg of the extracted DNA was digested separately by three DNA endonuclease combinations: NciI; HindIII and XhoI; SalI, XbaI and NdeI. RNase A was also added to each digestion mixture to remove any trace of contaminated RNA. After digestion, the digested products were purified by Qiagen PCR purification Kit. The three purified DNA sample were mixed together for the following blocking step.  2.Blocking of pre-existing strand break sites. Reaction mixture (40 μl) containing 4 μl of reaction buffer (NEBcutsmart buffer); 1 μl of Shrimp Alkaline Phosphatase (rSAP, NEB), 1 μg of template genomic DNA was incubated at 37 °C for 30 min to remove any pre-existing strand breaks with 3' end phosphate, then 70°C for 10min to inactivate rSAP. After cool down the reaction product to room temperature, add 2 μl of ddNTPs (2.5 mM each, TriLink) and 1 μl of DNA polymerase I (10 U, NEB), incubate at 37 °C for 40 min to block any pre-existing strand break sites. Then 1 μl of Shrimp Alkaline Phosphatase (rSAP, NEB) was added to the reaction mixture, incubate at 37 °C for another 30 min to degrade the excess ddNTPs. Reaction was terminated by heat-inactivation of rSAP and DNA polymerase I at 75 °C for 10 min. The reaction product was ready for one of the following nick creation or conversion procedure after de-salts by DyeEx column (QIAGEN)  3.Nick conversion of DNA PT sites by Ioline cleavage. 40 μl of the blocked DNA was added with 5 μl dibasic Sodium phosphate buffer (500mM, pH 9.0) and 2 μl of Iodine solution (0.1N, FLUKA). After incubate at 65°C for 5 minutes and cool down slowly to 4°C, the reaction product was purified by DyeEX column (QIAGEN) to remove salts and Iodine. The purified product was treated with rSAP by adding 5 μl of NEBcutsmart buffer and 1 μl of rSAP to remove any 3' end phosphate left from iodine cleavage.  After incubation at 37 °C for 20 min and 75 °C for another 10 min, the product was kept on ice for the following nick translation procedure with no additional purification.

value: iodine

tag: group


title: E14 cells, AC_6xPCR_no_ISceI_rep9

description: UMI-amplicon


title: mLN FRC

source_name: fibroblastic reticulum cells isolated from mesenteric lymph node (gp38+; CD31-)


title: EVS6_PYJ

city: Yangsan

first: Jae-Hwan

last: Choi

address: Kumo-ro 20, Beomo-ri, Mulgum-eup

department: Neurology

country: South Korea

institute: Pusan National University Yangsan Hospital

description: Gene expression data from Control

hyb_protocol: Approximately 5.5 μg of the labeled cDNAs were hybridized onto the Affymetrix GeneChip Array at 45℃ for 16 hours.

scan_protocol: After washing, the arrays were stained on a GeneChip Fluidics Station 450 and scanned on the GeneChip Scanner 3000.

data_processing:
Raw data were analyzed using Affymetrix GeneChip Command Console Software (AGCC). The raw data were summarized and normalized with Robust Multi-array Average (RMA) module implemented in Affymetrix Power Tools.
The differentially expressed genes (DEGs) between EVS and normal control samples were screened by fold-change (fc) filtering combined with student’s t-test. False discovery rate (FDR) was controlled by adjusted p-value using Benjamini-Hochberg algorithm. Genes were considered differentially expressed at |fold changes| > 1.5 and p-value < 0.05.

source_name: peripheral whole blood

label_protocol: Purified RNA sample was transcribed to double-strand cDNA using the GeneChip Whole Transcript (WT) Amplication kit. The sense cDNA was then fragmented and biotin-labeled with TdT (terminal deoxynucleotidyl transferase) using the GeneChip WT Terminal labeling kit.

extract_protocol: The samples were incubated for a minimum of 2 hours at room temperature (15-25℃) to achieve complete lysis of blood cells. And then, the samples were centrifuged for 10 minutes at 5000 x g using a swing-out rotor. The supernatant was decanted, and 4 mL of RNase-free water was added to the pellet. The samples were vortexed until the pellet was visibly dissolved, and centrifuged for 10 minutes at 5000 x g. After the entire supernatant was removed, the remaining pellet was resuspended in 350 μL of buffer BR1 and transferred to microcentrifuge tube, followed by automated RNA purification using QIAcube. The purity and quantity of RNA were measured using a NanoDrop ND-1000 (Thermo Fisher Scientiﬁc, Wilmington, DE, USA). The quality of RNA was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) with an RNA Integrity Number.

treatment_protocol: Peripheral whole blood (2.5 ml) was collected from the patients and seven healthy normal controls. The samples immediately placed into PAXgene Blood RNA Tubes (QIAGEN), and were stored under refrigerated conditions (2-8℃) before RNA extraction.

value: Whole blood

tag: disease

value: EVS with HV-DBN


title: pLN_exp2


title: EVS4_KHJ

description: Gene expression data from Control


title: EVS7_KMS

description: Gene expression data from Control


title: E14 cells, AC_6xPCR_no_ISceI_rep7

description: UMI-amplicon

value: AC


title: EVS5_KSY

description: Gene expression data from Control


title: mLN_exp1


title: 0mM-EndoIV-NT


title: E14 cells, AC_6xPCR_no_ISceI_rep6

description: UMI-amplicon


title: S87-NT-ctrl

description: Salmonella enterica serovar Cerro 87


title: pLN_exp1


title: S87-TdT-iodine

description: Salmonella enterica serovar Cerro 87


title: EVS2_JEJ

description: Gene expression data from AUV


title: 229_11_R5020+proclactin

city: Chuo-ku, Kobe

first: Bakhtiyor

last: Nosirov

state: Hyogo

address: Minatojima-minamimachi, 2-2-3

country: Japan

institute: BDR RIKEN, Japan

data_processing:
51 bp single end sequencing run with Illumina HiSeq 3000 platform and CASAVA was employed to generate the fastq file for each sample
RNA-seq FASTQ files were aligned to hg19 using TopHat (version 2.0.14) and only uniquely mapped reads were used for the further downstream analysis
summarizeOverlaps function (of GenomicAlignments package) in R/Bioconductor was utilized to count the reads that mapped to exonic regions only to generate a raw count matrix
DESeq2, whose default and recommended normalization method is relative log expression (RLE), was then utilized to identify differentially expressed genes (DEGs). The genes with log2FoldChange>=1, after rounding all log2FoldChange values to one decimal point, and adjusted p-value<0.05 were considered DEGs
Functional annotation of DEGs was performed using ClueGO (v2.5.4) plug-in of Cytoscape
Genome_build: hg19
Supplementary_files_format_and_content: read counts

source_name: T47D breast cancer cells

growth_protocol: Cell were maintained and passed in appropriate culture media, as described in the manuscript.

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

treatment_protocol: T47D breast cancer cells were treated with vehicle (water for prolactin and EtOH for R5020), 100 ng/ml prolactin, 10nM R5020, or both, for 6 hrs. RNA was isolated immediately following treatment.

value: breast cancer cells


title: 229_12_R5020+proclactin


title: EVS1_KSA

description: Gene expression data from AUV


title: E14 cells, AC_6xPCR_no_ISceI_rep4

description: UMI-amplicon


title: 229_10_R5020+proclactin


title: 229_09_Vehicle+proclactin


title: E14 cells, AC_6xPCR_no_ISceI_rep5

description: UMI-amplicon


title: E14 cells, AC_6xPCR_no_ISceI_rep2

description: UMI-amplicon


title: Control7_KKY

description: Gene expression data from AUV

value: Control


title: 229_08_Vehicle+proclactin


title: Kidney_DKD+enarodustat_rep7

city: Tokyo

first: Sho

last: Hasegawa

address: 7-3-1, Hongo, Bunkyo-ku

department: Division of Nephrology and Endocrinology

institute: University of Tokyo

description: Gene expression of renal cortex in DKD+enarodustat group

hyb_protocol: 0.6 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a Agilent fragmentation buffer and was hybridized at 65°C for 17 hr using Gene Expression Hybridization Kit.

scan_protocol: Microarray slides were scanned at 3 μm resolution using an Agilent G4900DA SureScan Microarray Scanner System.

data_processing: The intensity data of each individual hybridization were extracted and the quality was assessed with the Feature Extraction Software 11.5.1.1(Agilent). Raw data were processed with the R package limma in Bioconductor to perform background correction and data normalization using the quantile normalization method, and batch effects were removed by ComBat (batch 1; SAMPLE 1, 2, 6, 7, 13, 14, batch 2; SAMPLE 3, 4, 8, 9, 15, 16, batch 3; SAMPLE 5, 10, 11, 12, 17, 18, 19).

organism: Rattus norvegicus

source_name: Renal cortex, DKD+enarodustat, replicate 7

label_protocol: Agilent one-color Microarray-Based Gene Expression Analysis Low Input Quick Amp Labeling protocol

extract_protocol: Total RNA from kidney cortical tissue was isolated using GenElute Mammalian Total RNA Miniprep Kit (RTN70, Sigma-Aldrich). RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).

value: Renal cortex

value: Crl:CD (SD)

tag: gender

value: male

value: DKD+enarodustat


title: Control6_DKH

description: Gene expression data from AUV


title: EVS3_CHJ

description: Gene expression data from AUV


title: E14 cells, AC_6xPCR_no_ISceI_rep1

description: UMI-amplicon


title: Kidney_DKD+enarodustat_rep6

source_name: Renal cortex, DKD+enarodustat, replicate 6


title: Control5_GIS

description: Gene expression data from AUV


title: 229_06_R5020


title: E14 cells, AC_6xPCR_48h_rep3

description: UMI-amplicon


title: Control3_JYJ

description: Gene expression data from AUV


title: E14 cells, AC_6xPCR_48h_rep4

description: UMI-amplicon


title: Control4_JMH

description: Gene expression data from AUV


title: Kidney_DKD+enarodustat_rep4

source_name: Renal cortex, DKD+enarodustat, replicate 4


title: 229_05_R5020


title: Kidney_DKD+enarodustat_rep5

source_name: Renal cortex, DKD+enarodustat, replicate 5


title: E14 cells, AC_6xPCR_48h_rep2

description: UMI-amplicon


title: Kidney_DKD+enarodustat_rep3

source_name: Renal cortex, DKD+enarodustat, replicate 3


title: Control2_PKJ

description: Gene expression data from AUV


title: Kidney_DKD+enarodustat_rep2

source_name: Renal cortex, DKD+enarodustat, replicate 2


title: S87-TdT-ctrl

description: Salmonella enterica serovar Cerro 87

value: negative control

value: R2-TdT dependent


title: 229_04_R5020


title: 229_03_Vehicle


title: Control1_YJS

description: Gene expression data from AUV


title: E14 cells, AC_6xPCR_48h_rep1

description: UMI-amplicon

value: ES cells


title: 229_02_Vehicle


title: Kidney_DKD+enarodustat_rep1

source_name: Renal cortex, DKD+enarodustat, replicate 1


title: Core_fibrils_Passage T5_replicate B

city: Mumbai

first: Samir

last: Maji

address: Indian Institute of Technology Bombay, Powai

department: Biosciences and Bioengineering

country: India

institute: Indian Institute of Technology Bombay

description: Gene expression after treatment with treated with 30 µM of p53 core fibrils for passage T5, replicate B

hyb_protocol: Labeled cRNA sample were fragmented at 60ºC and hybridized on to a Agilent Human Gene  Expression Microarray 8X60K. Fragmentation of labeled cRNA and hybridization were done  using the Gene Expression Hybridization kit of (Agilent Technologies, In situ Hybridization kit,  Part Number 5190-0404). Hybridization was carried out in Agilent’s Surehyb Chambers at 65º C  for 16 hours. The hybridized slides were washed using Agilent Gene Expression wash buffers  (Agilent Technologies, Part Number 5188-5327).

scan_protocol: The hybridized slides were washed using Agilent Gene Expression wash buffers  (Agilent Technologies, Part Number 5188-5327) and scanned using the Agilent Microarray  Scanner (Agilent Technologies, Part Number G2600D).

data_processing:
Data extraction, dye normalization, and spatial derendering were done using default settings of the Agilent Feature Extractor and feature extracted raw data was analyzed using Agilent GeneSpring GX (v14.5) software.
Significant genes up regulated with fold change >=1  (logbase2) and down regulated with fold change <=-1 (logbase2) in the test samples with respect  to control sample were identified across the biological replicates.

source_name: MCF10A cells treated with p53 core fibrils and RNA was collected after 5 rounds of passaging (T5).

label_protocol: The samples for Gene expression were  labeled using Agilent Quick-Amp labeling Kit (p/n5190-0442). The total RNA were reverse  transcribed at 40°C using oligo dT primer tagged to a T7 polymerase promoter and converted to  double stranded cDNA. Synthesized double stranded cDNA were used as template for cRNA  generation. cRNA was generated by in vitro transcription and the dye Cy3 CTP(Agilent) was  incorporated during this step. The cDNA synthesis and in vitro transcription steps were carried  out at 40°C. Labeled cRNA was cleaned up using Qiagen RNeasy columns (Qiagen, Cat No:  74106) and quality assessed for yields and specific activity using the Nanodrop ND-1000.

growth_protocol: MCF10A cells were cultured in DMEM growth medium supplemented with 5% horse serum, 0.5 μg/ml hydrocortisone, 20 ng/ml hEGF, 10 μg/ml insulin and 1X Pen-Strep at 37 °C in a 5 % CO2 incubator.

extract_protocol: RNA was extracted using Qiagen Rneasy kit according to the manufacturers instructions.

treatment_protocol: For fibril treatment,DMEM containing 30 µM of p53 core fibrils were added alongwith other supplements were added and cells were incubated for 48 hours at 37 °C in a 5 % CO2 incubator.

value: breast

value: female

tag: age

value: Passage T5

value: p53 core fibrils


title: E14 cells, AC_6xPCR_24h_rep4

description: UMI-amplicon

data_processing:
[Processing inverse PCR reads]  The paired-end reads were pre-processed by custom Python scripts. The forward read consists of the barcode, a fixed 20 nucleotide watermark sequence used for identification, the NlaIII restriction site and some mouse genome sequence ligated to the NlaIII site. The reverse read consists of the last 25 nucleotides of pT2 and the mouse genome sequence at the insertion site of the transposon. We used seeq version 1.1 (https://github.com/ezorita/seeq) allowing up to three errors (mismatches and indels) to identify the watermark and isolate the barcode sequence. Reads were discarded if the watermark was not found or if the barcode was not between 14 and 24 nucleotides long. We removed the first 25 nucleotides of the reverse read and cut the sequence at the first NlaIII site, if any. The reads for which this sequence was shorter than 20 nucleotides were discarded. The genomic sequences thus obtained were mapped in the mouse genome release mm9 using the GEM mapper build 1.376 (Marco-Sola et al. 2012) with options `-q ignore --unique-mapping`. The reason for not using the more recent mouse release mm10 was that the chromatin from Juan et al. (2016) were only available in release mm9. After mapping, the barcodes were clustered using Starcode (Zorita, Cuscó, and Filion 2015) allowing up to two errors (options `-d2 --print-clusters`). This removes potential sequencing errors and consolidates the barcode sequences.   The barcodes were assigned to a genomic location using custom Python scripts. Unmapped genomic sequences were discarded and sequences mapping to multiple locations were flagged as such. For each barcode, we collected all the insertion sites that totalled at least 10% of the reads and we computed their diameter, equal to the maximum of their pairwise genomic distances (infinite for two sites on different chromosomes or if one of them maps to multiple locations). If the diameter was greater than 30 nucleotides, the barcode was discarded for being used in reporters mapping to different locations. Otherwise, the barcode was kept and its location was attributed to the most frequent insertion site (they are usually within 1-2 nucleotides of each other because of small mapping artefacts).
[Processing UMI-amplicon reads]  Paired-end reads were preprocessed using custom Python scripts. The forward read consists of the barcode, the watermark sequence and the right half of the second F segment . The reverse read consists of the UMI and the left half of the first F segment. Both reads extend the mid point of the F segment by three nucleotides. If the reporter is uncut or repaired by NHEJ, forward and reverse reads do not overlap. If the reporter is repaired by SSA, forward and reverse reads overlap because there is only one F segment. We can thus isolate the reads from reporters that went through SSA by ensuring that the nucleotides in the mismatch position are reverse-complement of each other in the forward and reverse reads. Thus, we used seeq with up to 10 errors to identify the half F segments and isolate the nucleotides in mismatch position on the forward and reverse reads, together with the barcode and the UMI. Barcodes and UMIs were clustered with Starcode allowing up to 2 errors (options `-d2 --print-clusters`) and the repair events were quantified for each barcode. The barcode–UMI pairs with only one read per run were discarded. After this operation, UMIs that were associated with more than one barcode were discarded. The remaining UMIs were classified as NHEJ or SSA as explained above, and those classified as SSA were further split into A/T or G/C. This provided for each barcode the full list of events reported by UMIs. Barcodes with more UMIs reporting NHEJ than SSA and those with only one UMI were removed. Barcodes that passed all these criteria in the control experiments without I-SceI induction were removed. The global proportion of remaining UMIs reporting A/T versus G/C was used a measure of repair bias.
Genome_build: mm9
Supplementary_files_format_and_content: mapped_repair.txt.gz


title: E14 cells, AC_6xPCR_24h_rep3

description: UMI-amplicon


title: LNCaP 10nM DHT 3

city: Gaithersburg

first: Kalpit

last: Shah

state: MD

address: 8717 Grovemont Cir

institute: DCEG

hyb_protocol: as per manufacturer's recommendatin (HTA-2.0)

scan_protocol: fluorescence intensity of the arrays was scanned using the Affymetrix Scanner and the raw data

data_processing: TAC console

source_name: LNCaP0nM DHT

growth_protocol: Grown for 72 hours in RPMI-1640 10% CSFBS media

extract_protocol: RNA was extracted using the RNeasy mini kit

treatment_protocol: Treated with either 10nM DHT, 10uM Casodex or DMSO

tag: tissue

value: Prostate

value: LNCaP

value: DHT


title: Kidney_DKD_rep7

description: Gene expression of renal cortex in DKD group

source_name: Renal cortex, DKD, replicate 7

value: DKD


title: 229_01_Vehicle

value: breast cancer cells


title: LNCaP DMSO 2

source_name: LNCaP DMSO

label_protocol: as per manufacturer's recommendatin (HTA-2.0)

value: DMSO


title: Kidney_DKD_rep6

source_name: Renal cortex, DKD, replicate 6

value: Renal cortex


title: Core_fibrils_Passage T1_replicate B

description: Gene expression after treatment with treated with 30 µM of p53 core fibrils for passage T0, replicate B

source_name: MCF10A cells treated with p53 core fibrils and RNA was collected after 48h at T1.

value: Passage T1


title: E14 cells, AC_6xPCR_24h_rep1

description: UMI-amplicon

source_name: ES cells

extract_protocol:
Genomic DNA from transfected cells was extracted using DNeasy Blood and Tissue Kit (Qiagen, #69504)
Inverse PCR: 10 μg genomic DNA from transfected mESCs were digested using 10 μl 10 U/μL NlaIII (NEB, #R0125S) in a 50 μL final volume for 3 hours at 37 °C. The reaction was heat-inactivated at 65 °C for 20 minutes. The reaction was diluted to a final volume of 1.8 mL in 1X T4 ligase buffer (Thermo Fisher Scientific, #EL0013) to favor self-ligation events, and ligation was carried out with 600 U of T4 ligase (Thermo Fisher Scientific, #EL0013) at 16°C overnight. After ligation, samples were precipitated by ethanol, pellets were resuspended in water and column-purified (QIAGEN, QIAquick PCR purification kit #28104) eluting with 100 uL EB.  Non circularized templates were eliminated by 2 h digestion at 37°C with Plasmid-safe DNAse (Epicentre, #E3101K), the enzyme was inactivated by heating for 30 minutes at 70°C. The product was column-purified (QIAGEN, QIAquick PCR purification kit #28104). The backbone of the TRIP reporters contains a I-CeuI site outside the transposable cassette, taking advantage of this, non integrated plasmids were cut by 2 h digestion at 37 °C with I-CeuI restriction enzyme (NEB, R0699S) in a total volume of 70 ul  followed by 20 minutes heat inactivation at 65 °C. All enzymatic reactions were carried out in the recommended manufacturer's buffer. For the first round of nested PCR, 10μL of I-CeuI-digestion products was mixed in 50 μL standard Phusion polymerase reaction mix (Thermo Fisher Scientific, F530S) in GC buffer, with 0.1 μM primers IR1,2 (annealing to IR/DR sequence). The cycling conditions were as follows: 98 °C for 5 min; 98°C for 20 s, 60°C for 1 min and 72°C for 5 min (1 cycle); 98°C for 20 s, 60°C for 1 min and 72°C for 2 min (20 cycles); and 72°C for 5 min. Products of the reaction were purified using Agencourt AMPure XP beads (Beckman Coulter, A63880), and eluted in 40 μL of water . For the second round of nested PCR, 37 μL of the products was diluted to 50 μL of standard Phusion polymerase reaction mix in GC buffer with 0.1 μM primer M1 (annealing to the Illumina PE1.0 primer) and one of indexing primers M2,3. The cycling conditions were as follows: 98°C for 2 min; 98°C for 20 s, 60°C for 1 min and 72°C for 1 min (10 cycles); and 72°C for 5 min. Primers M2,3 added the Illumina PE2.0 primer and one of indices to the amplicons. Products of the reaction were purified using Agencourt AMPure XP beads (Beckman Coulter, A63880), and eluted in 40 μL of water. PCR products ran as a smear on agarose gel. The smears were specific, because they failed to appear when the mESCs were not transfected. To eliminate I-SceI sites that were not cut during DSB induction and limit the size of PCR extension, 2 μg genomic DNA from mESCs (both, with DSB induction and control without the induction) were digested using 1 μl 100 U/μL XbaI (NEB, #R0145T) and 4 μl 5 U/μL I-SceI (NEB, #R0694L) in a 50 μL final volume for 3 hours at 37 °C. Digested DNA was column-purified (QIAGEN, QIAquick PCR purification kit #28104).
UMI-amplicons: To eliminate I-SceI sites that were not cut during DSB induction and limit the size of PCR extension, 2 μg genomic DNA from mESCs (both, with DSB induction and control without the induction) were digested using 1 μl 100 U/μL XbaI (NEB, #R0145T) and 4 μl 5 U/μL I-SceI (NEB, #R0694L) in a 50 μL final volume for 3 hours at 37 °C. Digested DNA was column-purified (QIAGEN, QIAquick PCR purification kit #28104).  UMI-LA 500 ng of genomic DNA obtained after I-SceI/XbaI digestion was used as a template in a 50 μL of Q5 DNA polymerase reaction mix (NEB, M0491S), using 50 nM of a UMI-containing primer U1 in the following cycling conditions: 98°C for 4 min; 98°C for 30s, 60°C for 1 min and 72°C for 1 min (50 cycles); and 72°C for 5 min. Products of linear amplification were purified using Agencourt AM Pure XP beads (Beckman Coulter, A63880), and eluted in 40 μL of water. Linear amplification was repeated 4 times for every sample to account for the technical variability.
UMI-PCR: 500 ng of genomic DNA obtained after I-SceI/XbaI digestion was used as a template in a 50 μL of Q5 DNA polymerase reaction mix (NEB, M0491S), using 50 nM of primers U1 and U2 in the following cycling conditions: 98°C for 1 min; 98°C for 20s, 60°C for 1 min and 72°C for 4 min (6 cycles); and 72°C for 10 min. Products of UMI-PCR were purified using Agencourt AM Pure XP beads (Beckman Coulter, A63880), and eluted in 40 μL of water. UMI-PCR was repeated 4 times for every sample to account for the technical variability.

value: AC


title: E14 cells, AC_6xPCR_no_ISceI_rep3

description: UMI-amplicon


title: E14 cells, AC_6xPCR_24h_rep2

description: UMI-amplicon


title: LNCaP 10uM bicul 2

source_name: LNCaP0uM bicul

value: Bicalutamide (Casodex)


title: Kidney_DKD_rep5

source_name: Renal cortex, DKD, replicate 5

value: Crl:CD (SD)

value: male


title: Human-EVs-6 [B6_EV]

city: Baltimore

first: Kenneth

middle: W.

last: Witwer

address: 733 N. Broadway

department: Department of Molecular and Comparative Pathobiology

institute: Johns Hopkins University School of Medicine

description:
IonXpressRNA_045_rawlib.basecaller_B6_EV
processed data file: Human_combined_raw_counts.xlsx

data_processing:
FASTQC run before and after adapter trimming. The reads were aligned to the reference transcriptomes in a sequential manner.   First, all reads were mapped to RNA species with low sequence complexity and/or high number of repeats: rRNA, tRNA, RN7S, snRNA, snoRNA, scaRNA, VT-RNA, Y-RNA as well as mitochondrial chromosome (mtRNA). All reads that did not map to the above RNAs were aligned to premiRNA reference, then to protein coding mRNA transcripts (pc) and long non-coding RNAs (lncRNAs). Please note that some % of reads always overlaps between pc and lncRNAs. The reads which did not map to premiRNA, protein coding (pc) transcripts and lncRNAs were aligned to the remaining transcriptome [other ncRNAs containing mostly pseudogenes and non-protein coding parts of mRNAs]. Finally, all reads which did not map to human transcriptome were aligned to the hg38 genome reference (rest_hg38).
The mapping was performed with bowtie2 default settings for all references except premiRNA. For premiRNA alignment I used sequential mapping with bowtie using settings with: 0 mismatch tolerance, then 1 mismatch tolerance and finally 2 mismatches tolerance.
Genome_build: Hg38
Supplementary_files_format_and_content: raw counts

source_name: Human-Evs

extract_protocol: Small RNA libraries were constructed using the Ion Total RNA-Seq Kit V2 (Life Technologies 4475936).

value: Brain

tag: tissue type

value: Parietal cortex

value: EV fraction from 10,000 x g supernatant after SEC and ultracentrifugation


title: LNCaP 10uM bicul 1