title: O2-G4

description: Library name: O2-G4


title: O2-G8

status: Public on Jun 15 2025

type: SRA

city: Murdoch

first: Viet

last: Dang

address: 90 South Street

country: Australia

institute: Murdoch University

description: Library name: O2-G8

library_source: genomic

data_processing:
Amplicon sequence variant (ASV) tables were constructed based on the DADA2 pipeline with default parameters. Read adapters were removed from raw read sequences using cutadapt, and read quality was inspected using FastQC. Chimeric sequences were removed from merged reads, and taxonomy was then assigned using the DADA2 "assignTaxonomy " function.
Assembly: metagenome
Supplementary files format and content: table files include ASV count and taxa
Supplementary files format and content: fasta files contain ASV sequences

molecule: genomic DNA

organism: soil metagenome

source_name: 0-10cm soil

extract_protocol:
DNeasy PowerSoil Kit (Qiagen Laboratories, Inc.)
Sequencing was carried out using Amplicon sequencing on Illumina MiSeq 2 × 250-bp PE platform

tag: tissue

value: 0-10cm soil


title: RIP

city: Harbin

first: Zheng

last: Chen

state: Heilongjiang

address: 92 Xidazhijie  Harbin Institute of Technology

country: China

institute: Harbin Institute of Technology

library_source: transcriptomic

data_processing:
Raw data (raw reads) of fastq format were firstly processed using fastp software. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low- quality reads from raw data. At the same time, Q20, Q30 and GC content of the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Index of the reference genome ( GRCm38/mm10) was built using BWA (v 0.7.12) and clean reads were aligned to the reference genome using BWA mem (v 0.7.12).
After mapping reads to the reference genome, we used the MACS2 (version 2.1.0) peak calling software to identify regions of IP enrichment over background. A q-value threshold of 0.05 was used for all data sets. After peak calling, the distribution of chromosome distribution, peak width, fold enrichment, significant level and peak summit number per peak were all displayed. The interaction between Intracellular RNA and protein binding were not random, while they show some specific sequence preference.
Homer（v4.9.1） was used to detect the denovo sequence motif and the matched known motifs. Peak related genes can be confirmed by Peak Annotator.
GO enrichment analysis was implemented by the GOseq R package, in which gene length bias was corrected. GO terms with corrected P-value less than 0.05 were considered significantly enriched by peak related genes. KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other highthrough put experimental technologies (http://www.genome.jp/kegg/). We used KOBAS (3.0) software to test the statistical enrichment of peak related genes in KEGG pathways.
Assembly: mouse reference genome( GRCm38/mm10)
Supplementary files format and content: Processed data files; excel; RIP_peaks_table

molecule: total RNA

organism: Mus musculus

source_name: primary hepatocytes

growth_protocol: Primary hepatocytes were isolated from C57BL/6 WT mice by liver perfusion with type II collagenase (Worthington Biochem, Lakewood, NJ) and cultured at 37°C and 5% CO2 in RPMI 1640 medium supplemented with 3% FBS.

extract_protocol:
Cells were harvested in a lysis buffer(100 mM KCl, 5 mM MgCl2, 10 mM HEPES (pH 7.0), 0.5% NP40, 1 mM DTT, 100 units/mL RNaseOut) that was made in DEPC H2O. 10% of cell lysate was used for input. The left cell lysate was immunoprecipitated with 100 μL anti-FLAG M2 magnetic beads (M8823, Millipore) at 4 ℃ for 2 hours. The immunoprecipitated samples were washed using lysis buffer for 4 times. Total RNA was extracted from the immunoprecipitated and input samples using the TriPure Isolation Reagent (Roche, Mannheim, Germany). Message RNA was purified by using  Hieff NGS® mRNA Isolation Master Kit (12603, Yeasen Biotechnology).
The fragment distribution and concentration of RNAs（after immunoprecipitation） were detected using an Agilent 2100 bioanalyzer (Agilent) and simpliNano spectrophotometer (GE Healthcare). Then, immunoprecipitated RNA or Input was used for library construction with NEB NextR Ultra™ RNA Library Prep Kit (New England Biolabs). Library quality was assessed on the Agilent Bioanalyzer 2100 system.
The libraries were sequenced on an Illumina Novaseq 6000 platform with a paired-end read length of 150 bp according to the standard protocols.

treatment_protocol: Primary hepatocytes from C57BL/6 WT mice were infected with Ad-FLAG-YTHDC1 adenoviruses overnight.

tag: cell type

value: primary hepatocytes

tag: treatment

value: Primary hepatocytes were infected with Ad-FLAG-YTHDC1 adenovirus overnight. RNA immunoprecipitation sequence (RIP-seq) assay was performed.


title: O1-G4

description: Library name: O1-G4


title: U3179MG, scRNAseq

city: Uppsala

first: Ida

last: Larsson

address: Dag Hammarskjölds Väg 20

country: Sweden

institute: Uppsala University

description: 10X Genomics

library_source: transcriptomic single cell

data_processing:
Demultiplexing, counting, and alignment to the human (GRCh38) reference genome were performed using Cell Ranger 3.0.2 using default parameters.
Assembly: GRCh38
Supplementary files format and content: Tab-separated values files and matrix files.

organism: Homo sapiens

growth_protocol: Patient-derived cells cultured in Neural Stem Cell Media (cells were seeded on 1% laminin-coated flasks and maintained in serum-free neural stem cell medium with B-27 and N2 supplements, as well as EGF and FGF growth factors). As well as, patient-derived cells isolated from NMRI nude (NMRI-Foxn1 nu/nu) mice or Hsd:Athymic nude-Foxn1.

extract_protocol:
Upon the experimental endpoint, mouse brains were harvested into cold HBSS buffer containing 1% Pen/Strep, 0.6% glucose, and 25nM HEPES. Then, the brain was sliced using a 1mm coronal section matrix, cut into about 1-2 mm pieces using a surgical blade, and dissociated into single cells using the Tumor Dissociation kit, human (Miltenyi, #130-095-929), used in combination with the Mouse Cell Depletion Kit (Miltenyi, #130-104-694) according to the manufacturer’s protocols. Red blood cells were removed using the Red Blood Cell Lysis Solution (Miltenyi, #130-094-183
The generation of single-cell RNA sequencing libraries followed the manufacturer’s guidelines, utilizing the Chromium Single Cell 3’ Library and Gel Bead Kit v2, v3, and v3.1 (analysis of KO-cells) (10x Genomics, Pleasanton, CA). Cryo-preserved cells underwent washing and re-suspension in 0.1% BSA in PBS just before loading onto a Chromium Single Cell B Chip (10X Genomics) with the aim of capturing 10,000 cells.

tag: cell line

value: Patient-derived GBM cells

tag: Sex

value: Male


title: Petunia inflata, young bud, 12 weeks

city: Davis

first: Blake

last: Meyers

state: CA

address: 451 Health Sciences Drive

department: Department of Plant Sciences

country: USA

institute: UC  Davis Genome Center

description:
Library name: PinfW-05_i1-20
sRNA library from Petunia inflata. These libraries were generated from 4-5 large (3-4 mm) buds at plant age 12 weeks (Plant IDs 6-10)

data_processing:
Raw small RNA sequence data was trimmed on quality at a cut-off value of > 0.05, a maximum of 2 ambiguities, adapters, and length between 15-34 nt, using the CLC Genomic Workbench (version 7.0; http://www.clcbio.com). Small RNA contaminants were filtered-out on the basis of a BLAST search 21 against a database including tRNAs from Arabidopsis thaliana (Ath) (http://lowelab.ucsc.edu/GtRNAdb/ Athal) and Rfam 11.0 (http://www.sanger.ac.uk; subset RF00005), small subunit rRNAs from Rfam (subsets RF00001;5S rRNA and RF00002;5_8S_rRNA), mtDNAs from the tomato mitochondrial genome (http://www.mitochondrialgenome.org) and GenBank: Ath, Solanum lycopersicum (Sly), Nicotiana tabacum (Nta), and Vitis vinifera (Vvi), and cpDNAs from GenBank: Ath, Sly, Solanum tuberosum (Stu), Nta, Glycine max (Gma), Medicago truncatula (Mtr) and Vvi, using an identity of ≥ 80 %.
Assembly: P. axillaris N and P. inflata S6.
Supplementary files format and content: tab-delimited file in which each line contains a unique RNA sequence and the number of times that sequence appears in the library

organism: Petunia integrifolia subsp. inflata

source_name: buds

extract_protocol:
Total RNA was isolated by a standard method using TRIzol® Reagent (Life Technologies), including a second precipitation step to increase RNA purity, and using 20 minutes centrifugation, 14.000 rpm, to optimize small RNA recovery. Samples were stored in the second precipitation, in 0.3 M Sodium Acetate (NaAc)/70 % Ethanol (EtOH), at -80 °C, upon library preparation. RNA was suspended in 10-30 μl DEPC-MQ to a final concentration of ~ 1 μg/μl, and checked on agarose for a 25S to 18S ribosomal peak ratio of > 4:5 to be used for library preparation. Small RNA libraries were prepared from 1.2 μg total RNA, using the Illumina compatible NextFlex™ Small RNA Sequencing Kit (version 1; Bioo Scientific; Sanbio BV, Uden, The Netherlands; Cat. no. 5132) and accompanying Small RNA Barcodes Primers (Cat. no. 5133). This protocol included an 18 h long 3’-adapter ligation of pre-adenylated oligonucleotides, with a modified ligase, to optimize binding to plant miRNAs and other small RNAs that contain 2’-O-methylated 3’-ends (Munafó and Robb, 2010; McCormick et al., 2011). After the 5'-adapter ligation, the recommended purification step was included, using the RNA Clean and Concentrator Kit (Zymo Research; BaseClear, Leiden, The Netherlands; Cat. no. R1015). We amplified the cDNAs by 14-16 PCR cycli. The ~ 150 nt long small RNA libraries were isolated from 3 % NuSieveTM (Lonza, Basel, Switzerland)/1 % agarose gels, and extracted by the QIAquick Gel Extraction Kit (Qiagen Benelux BV, Venlo, The Netherlands; Cat. no. 28706). Libraries were eluted in 40μl pre-warmed buffer of the Kit, and precipitated in NaAc/EtOH, at -20°C, after addition of 1 μl co-precipitant of the NextFlex Kit.
After re-suspension of the small RNA libraries in 10 μl MQ, they were sent to BaseClear (Leiden, The Netherlands) for Illumina HiSeq 2500 sequencing. Quality Control included a BioAnalyzer measurement (Agilent Technologies) and qPCR, on which basis the samples were pooled in equal amounts to 18 samples/lane in order to provide a minimum of ~ 5 Million Reads/library. Sequence data was de-multiplexed and reads delivered as '.fastq' files together with the run-statistics.

value: buds

tag: genotype

tag: time point

value: 12 weeks


title: Petunia axillaris, young bud for 18 weeks

description:
Library name: PaxW-05_A3-24
sRNA library from Petunia axillaris. These libraries were generated from 4-5 young (<3 mm) buds at plant age 18 weeks (Plant IDs 11-15)

organism: Petunia axillaris

value: PaxiN

value: 18 weeks


title: Input

value: Primary hepatocytes were infected with Ad-FLAG-YTHDC1 adenovirus overnight.


title: Y1HKO

data_processing:
Raw data (raw reads) of fastq format were firstly processed using fastp (version 0.20.0). In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low-quality reads from raw data. At the same time, Q20, Q30 and GC content of the clean data were calculated. All the downstream analyses were based on the clean data.
Reference genome and gene annotation files were downloaded from genome website directly. Index of the reference genome was built using BWA(v0.7.12) and clean reads were aligned to the reference genome using BWA mem. Reads that were derived from mitochondrion DNA and chloroplast DNA were discarded.These reads were then filtered for high quality (MAPQ ≥ 13),we also removed reads that were not properly paired and with PCR duplicates. Only uniquely mapped(MAPQ ≥ 13) and de-duplicated reads were used for further analysis.
All peak calling was performed with MACS2 (version 2.1.0) using ‘macs2 -q 0.05 -f AUTO --call-summits --nomodel --shift -100 --extsize 200 --keep-dup all’. By default, peaks with q-value threshold of 0.05 was used for all data sets.
Peaks were adjusted to the same size (500 bp) centered peak summits and motif discoveries of these loci sequences were performed using findMotifsGenome.pl program in HOMER v4.11 software with ‘-len 8,10,12,14 -gc -size given -homer2 -dumpFasta’.
The position of peak summit around transcript start sites of genes can predict the interaction sites between protein and gene. ChIPseeker (Yu et al., 2015) was used to retrieve the nearest genes around the peak and annotate genomic region of the peak. Peak-related genes can be confirmed by ChIPseeker, and then Gene Ontology (GO) enrichment analysis was performed to identify the function enrichment results. GO enrichment analysis was implemented by the GOseq R package, in which gene length bias was corrected. GO terms with corrected P-value less than 0.05 were considered significantly enriched by peak-related genes. KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies (http://www.genome.jp/kegg/). We used KOBAS software to test the statistical enrichment of peak related genes in KEGG pathways.
Assembly: mouse reference genome(grcm38_p6)
Supplementary files format and content: Processed data files; excel; Y1HKO_vs_Y1FF_peak_compare_table

source_name: liver

extract_protocol:
Each liver sample was pooled from four Ythdc1-HKO and Ythdc1flox/flox mice at 9 weeks old, respectively.Nuclei was extracted from liver samples.
Briefly, nuclei was extracted from samples, and the nuclei pellet was resuspended in the Tn5 transposase reaction mix. The transposition reaction was incubated at 37℃ for 30 min. Equimolar Adapter1 and Adatper 2 were added after transposition, PCR was then performed to amplify the library. After the PCR reaction, libraries were purified with the AMPure beads and library quality was assessed with Qubit.
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufactuer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina platform and 150 bp paired-end reads were generated.

value: liver


title: O2-G9Z10

description: Library name: O2-G9Z10


title: Petunia axillaris, young bud, 18 weeks

description:
Library name: PaxW-05_A2-23
sRNA library from Petunia axillaris. These libraries were generated from 4-5 young (<3 mm) buds at plant age 18 weeks (Plant IDs 11-15)


title: O2-L1

description: Library name: O2-L1


title: U3031MG, PDX, scRNAseq

description: 10X Genomics

source_name: Patient derived cells isolated from mouse PDX (Brain)

value: U3031MG PDX

value: Patient-derived GBM xenografted cells

value: Female


title: U3031MG, scRNAseq

description: 10X Genomics


title: Y1FF


title: U3220MG, PDX, replicate 2, scRNAseq

description: 10X Genomics

value: Patient derived cells isolated from mouse PDX (Brain)

value: U3220MG PDX


title: O2-L10

description: Library name: O2-L10


title: Petunia axillaris, larger bud,18 weeks

description:
Library name: PaxW-05_A1-25
sRNA library from Petunia axillaris. These libraries were generated from 4-5 young (<3 mm) buds at plant age 18 weeks (Plant IDs 11-15)


title: P21_Treatment_Sun1-GFP_multiome

city: Piscataway

first: Tulsi

last: Patel

state: New Jersey

address: 683 Hoes Lane West

department: Neuroscience and Cell Biology

institute: Rutgers University

description: 10x Genomics Multiome

library_source: other

data_processing:
RSEM alignment for bulk RNAseq data
CellRanger for 10x genomics data
Assembly: mm10
Supplementary files format and content: genes, counts, fpkm, tpm for bulk data.
Supplementary files format and content: Input needed for Seurat for 10x genomics data
Library strategy: Multiome

molecule: other

source_name: Spinal Cord

extract_protocol:
protocol for nuclei purification + FACs in Patel et al., 2022 Nature Neuroscience
SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian for bulk RNAseq, 10x Genomics kit for multiome

treatment_protocol: AAV-ChatEn-Isl1 + AAV-ChatEn-Lhx3 were injected ICV at P1

value: Spinal Cord

value: AAV-Isl1 + AAV-Lhx3


title: U3220MG, PDX, replicate 1, scRNAseq

description: 10X Genomics


title: O2-L11

description: Library name: O2-L11

value: 0-10cm soil


title: P21_Control_Sun1-GFP_multiome

description: 10x Genomics Multiome

value: no treatment


title: Petunia axillaris, large bud, 12 weeks

description:
Library name: PaxW-05_A1-12
sRNA library from Petunia axillaris. These libraries were generated from 4-5 young (<3 mm) buds at plant age 12 weeks (Plant IDs 11-15)


title: U3220MG, scRNAseq

description: 10X Genomics

value: Patient-derived GBM cells


title: O2-L2

description: Library name: O2-L2


title: D60_2

data_processing:
The image data measured by the high-throughput sequencer are converted into  sequence data (reads) by CASAVA base recognition. Raw data (raw reads) of fastq  format were firstly processed through in-house perl scripts. In this step, clean data  (clean reads) were obtained by removing reads containing adapter, reads containing N  base and low quality reads from raw data. At the same time, Q20, Q30 and GC  content the clean data were calculated. All the downstream analyses were based on  the clean data with high quality.
Reference genome and gene model annotation files were downloaded from  genome website directly. Index of the reference genome was built using Hisat2  (v2.0.5) and paired-end clean reads were aligned to the mouse reference genome(GRCm38/mm10) using Hisat2 (v2.0.5). We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.
featureCounts (v1.5.0-p3) was used to count the reads numbers mapped to each  gene. And then FPKM of each gene was calculated based on the length of the gene  and reads count mapped to this gene. FPKM, expected number of Fragments Per  Kilobase of transcript sequence per Millions base pairs sequenced, considers the  effect of sequencing depth and gene length for the reads count at the same time, and is  currently the most commonly used method for estimating gene expression levels.
Differential expression analysis of two  conditions/groups (two biological replicates per condition) was performed using the  DESeq2 R package (1.20.0). DESeq2 provide statistical routines for determining  differential expression in digital gene expression data using a model based on the  negative binomial distribution. The resulting P-values were adjusted using the  Benjamini and Hochberg’s approach for controlling the false discovery rate .  padj<=0.05 and |log2(foldchange)| >= 0 were set as the threshold for significantly  differential expression.
Assembly: mouse reference genome(GRCm38/mm10)
Supplementary files format and content: Processed data files; excel; fpkm of each gene

extract_protocol:
Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany) from livers of WT mice at day 1, day 10, day 20 and day 60 after birth. Three independent biological replicates for each group were used for RNA-seq.
mRNA was purified from total RNA by using poly-T oligo-attached magnetic beads.  Fragmentation was carried out using divalent cations under elevated temperature in  First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using  random hexamer primer and M-MuLV Reverse Transcriptase, then use RNaseH to  degrade the RNA. Second strand cDNA synthesis was subsequently performed using  DNA Polymerase I and dNTP. Remaining overhangs were converted into blunt ends  via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA  fragments, Adaptor with hairpin loop structure were ligated to prepare for  hybridization. In order to select cDNA fragments of preferentially 370~420 bp in  length, the library fragments were purified with AMPure XP system (Beckman  Coulter, Beverly, USA). Then PCR amplification, the PCR product was purified by  AMPure XP beads, and the library was finally obtained.
After the library is qualified, the different libraries are pooling according to the  effective concentration and the target amount of data off the machine, then being  sequenced by the Illumina NovaSeq 6000. The end reading of 150bp pairing is  generated. The basic principle of sequencing is to synthesize and sequence at the same  time (Sequencing by Synthesis). Four fluorescent labeled dNTP, DNA polymerase  and splice primers were added to the sequenced flowcell and amplified. When the  sequence cluster extends the complementary chain, each dNTP labeled by  fluorescence can release the corresponding fluorescence. The sequencer captures the  fluorescence signal and converts the optical signal into the sequencing peak by  computer software, so as to obtain the sequence information of the fragment to be  tested.

value: WT

tag: straiin

value: day 60 after birth


title: jacP16_cd6cranial_22

city: La Jolla

first: Ceri

middle: J

last: Weber

address: 9500 Gilman Dr

institute: University of California San Diego

description: Library name: Jac8

data_processing:
Samples were demultiplexed using bcl2fastq Conversion Software
Quality control analysis and trimming of raw fastq files were performed using Trim Galore, a wrapper around Cutadapt and FastQC.
Trimmed reads were mapped to TOGA-annotated one-to-one and one-to-zero transcripts using STAR and read counts per gene were acquired by “--quantMode GeneCounts” option.
STAR aligned gene counts were used to perform differential expression analysis between mouse and jerboa TV1 and TV6 with DESeq2.
Assembly: Mus musculus (mm10) and Jaculus jaculus (mJacJac1.mat.Y.cur)
Supplementary files format and content: SR50 files are the raw count matrices

organism: Jaculus jaculus

source_name: Tail vertebra 6 cranial growth cartilage

growth_protocol: Mouse pups were raised to postnatal day 7 and jerboa pups were raised to postnatal day 16

extract_protocol:
Three cranial growth plates per vertebral identity per species was pooled into RNAlater (Invitrogen) and equilibrated overnight at 4˚C. Samples were removed from RNAlater and snap frozen in liquid nitrogen the next day and stored at -80˚C or immediately processed for RNA extraction. Tissue was disrupted over liquid nitrogen using a mortar and pestel, then homogonized using Qiagen QIAshredder system. RNA was extracted using the Qiagen RNAeasy Micro kit with on-column DNA-digest
UCSD IGM used Illumina mRNA Stranded Library kits to prepare libraries from samples with Agilent Tapestation 4200 RIN scores >8

treatment_protocol: Tails were incubated in Proteinase K to remove connective tissues. Cranial growth cartilages were dissected from mouse and jerboa tail vertebra 1 and tail vertebra 6 in ice cold PBS.

tag: time

value: postnatal day 16


title: U3054MG, PDX, replicate 1, scRNAseq

description: 10X Genomics

value: U3054MG PDX


title: Petunia axillaris, young bud, 9 weeks

description:
Library name: PaxW-05_A0-34
sRNA library from Petunia axillaris. These libraries were generated from 4-5 young (<3 mm) buds at plant age 9 weeks (Plant IDs 11-15)

value: buds

value: PaxiN

value: 9 weeks


title: jacP16_cd6cranial_12

description: Library name: Jac7

value: Tail vertebra 6 cranial growth cartilage


title: O2-L4

description: Library name: O2-L4


title: D60_3

value: liver


title: U3054MG, scRNAseq

description: 10X Genomics

value: Female


title: D60_1


title: O2-L3

description: Library name: O2-L3


title: Petunia axillaris, young bud, 12 weeks

description:
Library name: PaxW-05_A2-14
sRNA library from Petunia axillaris. These libraries were generated from 4-5 young (<3 mm) buds at plant age 12 weeks (Plant IDs 11-15)

value: 12 weeks


title: D20_3

value: day 20 after birth


title: jacP16_cd6cranial_10

description: Library name: Jac6


title: U3013MG, PDX, replicate 2, scRNAseq

description: 10X Genomics

value: Patient derived cells isolated from mouse PDX (Brain)

value: U3013MG PDX

value: Patient-derived GBM xenografted cells


title: U3013MG, PDX, replicate 1, scRNAseq

description: 10X Genomics


title: jacP16_cd6cranial_8

description: Library name: Jac5


title: D20_2


title: D20_1


title: jacP16_cd1cranial_21

description: Library name: Jac4

source_name: Tail vertebra 1 cranial growth cartilage


title: O2-L7

description: Library name: O2-L7

value: 0-10cm soil


title: jacP16_cd1cranial_20

description: Library name: Jac3

value: Tail vertebra 1 cranial growth cartilage


title: O1-G6

description: Library name: O1-G6


title: D10_3

value: WT

value: day 10 after birth


title: O2-L8

description: Library name: O2-L8


title: LIB5455514_SAM24414654_S7_L001

city: South San Francisco

first: Marc

last: Hafner

address: Building 45-1, 1 DNA Way

department: Oncology Bioinformatics

institute: Genentech

description:
SAM24414654_LIB5455514
NovaSeq4

data_processing:
CMO data was processed using CellRanger and scRNA-Seq was processed using STARsolo with standard parameters.
Cumulus workflow with default settings was used for demultiplexing
Assembly: GRCh38 (GENCODE v32/Ensembl 98)

source_name: Colon

growth_protocol: The cells were seeded to 6-well plates. All samples were treated with compound combinations starting at different days and harvested together. Upon sample collection, cells were trypsinized from the plate and hashed by using the cholesterol modified oligo (CMO) following the MULTI-seq protocol

extract_protocol:
Cells from each sample were washed and re-suspended in 180µl of PBS+1%BSA and transferred to a round-bottom 96-well plate. Then 20µl of each anchor-barcode mix was added to the suspension for a final concentration of 2µM. After the incubation for 15 minutes at room temperature, 20µl of co-anchor was added to each sample (final concentration of 2µM) and incubate for another 15 minutes at RT. Cells were then washed by 500µl of cold PBS+1% BSA twice before pooling together.
The library construction was done following the manufacturer’s manual with a few modifications to accommodate the MULTI-seq protocol: (1) during the cDNA amplification 1µl of the following primer (10µM) was added to the master mix, 5’-CTTGGCACCCGAGAATTCC-3’; (2) the supernatant from the 0.6X SPRI beads cleanup for cDNA was kept and further cleaned up by using 2.0X SPRI beads; (3) the CMO libraries were made by performing 8 cycles of the indexing PCR using the supernatant. The transcriptome and CMO libraries were then sequenced by using either the Illumina NovaSeq or NextSeq machines

tag: library name

value: Colon

value: CRC line

tag: sample name


title: U3054MG, PDX, replicate 2, scRNAseq

description: 10X Genomics

value: U3054MG PDX


title: U3013MG, scRNAseq

description: 10X Genomics

value: Patient-derived GBM cells


title: D10_2


title: jacP16_cd1cranial_19

description: Library name: Jac2

value: postnatal day 16


title: LIB5455513_SAM24414654_S8_L001

description:
SAM24414654_LIB5455513
NovaSeq4


title: O2-L5Z6

description: Library name: O2-L5Z6


title: O2-L9

description: Library name: O2-L9


title: O2-L6

description: Library name: O2-L6


title: jacP16_cd1cranial_9

description: Library name: Jac1


title: D10_1


title: LIB5455512_SAM24414654_S5_L001

description:
SAM24414654_LIB5455512
NovaSeq4


title: LIB5455517_SAM24414655_S4_L001

description:
SAM24414655_LIB5455517
NovaSeq4


title: D1_3

value: day 1 after birth


title: LIB5455516_SAM24414655_S9_L001

description:
SAM24414655_LIB5455516
NovaSeq4


title: O2-Z1

description: Library name: O2-Z1


title: O2-Z2

description: Library name: O2-Z2


title: O2-Z11

description: Library name: O2-Z11


title: [PRO-Seq] K562 cells, LP recombination, e1 only mGATA1 (no e2), rep1

city: Ithaca

first: Zhou

last: Zhou

state: New York

address: 526 Campus Rd, 417 Biotechnology Building

department: Molecular Biology and Genetics

institute: Cornell University

description:
Library name: Sample 13
"merge" bw files are merged from rep1 and rep2 bw

data_processing:
For all NGS sequencing data, the quality of FASTQ files was first accessed using FastQC, and the Illumina sequencing adaptors were trimmed using fastp.
To enable accurate alignment of sequencing reads to the eNMU locus in the recombinant cell lines, we built a custom genome for each recombinant TF motif mutant using the reform command line tool. Each custom genome incorporated the exact mutant sequence flanked by Bxb1 recombination sites. For PRO-seq data analysis, rRNA read removal, alignment, and deduplication (with UMI-tools) were performed using the published bash script at https://github.com/JAJ256/PROseq_alignment.sh.
Unnormalized 3'-end bigwigs files for PRO-seq were first generated from bam alignment files by PINTS using the option “pints_visualizer -e R1_5 --reverse-complement”. To calculate scaling factors for PRO-seq data, a collapsed list of all GENCODE v46 transcripts was first generated using the reduceByGene() function in the BRGenomics R package. The unnormalized bigwig files were loaded into Rstudio, and a DESeq2 object was generated for the combined list of all PRO-seq samples using the getDESeqDataSet() function in BRGenomics, using the collapsed transcript list to specify genomic regions of interest. Scaling factors calculated by DESeq2 were applied to each bigwig file using the applyNFsGRanges() function in BRGenomics before data export. Unnormalized 5'-end bigwigs files for PRO-seq were generated from bam alignment files by PINTS using the option “pints_visualizer -e R2_5” and normalized using the same scaling factors calculated from the 3'-end bigwigs files. Normalized bigwig files of two biological replicates were merged using kentUtils of the UCSC Genome Browser for visualization.
PROseq bigwig files are all at single basepair resolution, with 3'-end read position files labled as "PRO_3p_XXX" and 5'-end read position files labled as "PRO_5p_XXX".
Assembly: hg38
Supplementary files format and content: bigwig
Library strategy: PRO-Seq

molecule: nuclear RNA

growth_protocol: All the K562 lines were cultured in RPMI 1640 media supplemented with GlutaMAX (Gibco) and 10% heat-inactivated FBS (Avantor) at 37°C with 5% CO2 in a humidified sterile incubator. Cell density was maintained between 0.1 ~ 1 × 10⁶ cells/mL, and mycoplasma testing was performed routinely.

extract_protocol:
All buffer compositions and reaction conditions followed the published protocol of Mahat and Kwak et al. (2016) unless otherwise specified. Briefly, 5 million K562 cells were mixed with 250,000 Drosophila S2 cells (5% spike-in), pelleted at 1000 r.c.f. for 5 min at 4°C, washed once with ice-cold PBS, resuspended with ice-cold permeabilization buffer at a density of 1 million cells/mL, and incubated on ice for 5 min. Cells were washed twice with the same volume of ice-cold permeabilization buffer, and resuspended in 100 μL storage buffer before immediate nuclear run-on or flash-freezing in liquid nitrogen for long-term storage at –80°C. Prior to the run-on reaction, 40 μL Dynabeads MyOne Streptavidin C1 beads (Thermo Fisher) per sample were pre-washed sequentially with Hydrolysis Buffer (0.1N NaOH + 50 mM NaCl), High Salt Wash Buffer, and Binding Buffer. Pre-washed beads were resuspended in 60 μL Binding Buffer per sample. The nuclear run-on reaction was performed at 37°C for 5 min with a final concentration of 20 µM each of Biotin-11-CTP, Biotin-11-UTP, ATP, and GTP. Following RNA extraction by Trizol LS (Invitrogen) and RNA fragmentation by base hydrolysis, 30 μL pre-washed C1 beads and 30 μL Binding Buffer were added to the ~60 μL RNA sample, and bead binding was performed at room temperature for 20 min on a rotational device. Beads were then washed twice using 500 μL High Salt Wash Buffer and once using 500 μL Low Salt Wash Buffer, with tube swap after each wash. Biotinylated RNA was eluted from beads by Trizol extraction, and 3' RNA adaptor ligation was performed in a total volume of 20 μL with 5 µM final adaptor concentration and 2 μL T4 RNA Ligase I, High Concentration (NEB). The reaction was incubated at 20°C for 4 h and held at 4°C overnight. On the next day, 50 μL Binding buffer and 30 μL pre-washed C1 beads were added to the reaction, and another round of bead binding and bead washing was performed as described above. Subsequent 5' enzymatic modifications of RNA were performed on beads with a reaction volume of 20 μL assuming 1 μL bead volume: 5' decapping reaction involved 1 μL RppH (NEB) and 1-h incubation at 37°C; 5' hydroxyl repair involved 1 μL T4 PNK (NEB) and 1-h incubation at 37°C. Beads were then washed once with 300 μL Binding Buffer, and 5' RNA adaptor ligation was performed on beads in a total volume of 20 μL with 5 µM final adaptor concentration and 2 μL T4 RNA Ligase I, High Concentration (NEB), incubated at room temperature for 1 h on a rotational device. Beads were washed twice using 500 μL High Salt Wash Buffer and once using 500 μL Low Salt Wash Buffer, with tube swap after each wash. RNA was eluted from beads by Trizol extraction and resuspended in 13 μL RT resuspension mix (8 μL DEPC H2O, 4 μL of 10 μM Illumina RP1 primer, 1 μL of 10 mM dNTPs). RNA was denatured at 65°C for 5 min and snap cooled on ice, and 7 μL RT master mix was added to the sample (4 μL of 5× RT Buffer, 1 μL of 100 mM DTT, 1 μL Invitrogen SUPERase·In RNase Inhibitor, 1 μL Thermo Scientific Maxima H Minus Reverse Transcriptase). RT reaction program was: 50°C for 30 min, 65°C for 15 min, 85°C for 5 min, hold at 4°C. The resulting cDNA was diluted with an equal volume of DEPC H2O.
A test amplification was performed on 1:4 serial dilutions of 2 μL cDNA sample and run on 6% native PAGE TBE gel to determine the optimal PCR cycle number (N). Final amplification was performed in 100 μL volume (32.5 μL DEPC H2O, 20 μL of 5× Phusion HF Buffer, 20 μL of 5 M betaine, 2.5 μL of 10 µM Illumina RP1 primer, 2.5 μL of 10 µM Illumina indexing RPI-n primer, 2.5 μL of 10 mM dNTPs, 1 μL homemade Phusion polymerase, 19 μL cDNA sample). PCR program was: initial denaturation 95°C for 2 min; 5 cycles of 95°C for 30 s, 56°C for 30 s, 72°C for 30 s; N cycles of 95°C for 30 s, 65°C for 30 s, 72°C for 30 s; final extension 72°C for 5 min. PCR products were sequentially purified using the MinElute PCR purification kit (Qiagen) and homebrew SPRI beads (1.5× bead ratio) to remove all unused primers. DNA concentration was measured by the Qubit dsDNA High Sensitivity assay (Thermo Fisher). The libraries were pooled for sequencing on the Element Biosciences AVITI (2 × 80 bp paired-end sequencing) or Illumina NovaSeq 6000/X Plus platform (2 × 150 bp paired-end sequencing). Note that the 3' and 5' RNA adaptors contain a 6-nt unique molecular identifier (UMI) to enable accurate identification of PCR duplicates in downstream bioinformatic analysis. The complete sequences of the adaptors can be found in Judd et al., 2020.

value: erythroleukemia


title: [PRO-Seq] K562 cells, LP recombination, e1 only mGATA1 (no e2), rep2

description: Library name: Sample 14


title: D1_1

value: liver


title: LIB5455515_SAM24414655_S6_L001

description:
SAM24414655_LIB5455515
NovaSeq4


title: O2-Z3

description: Library name: O2-Z3


title: O2-Z4

description: Library name: O2-Z4


title: HKO_2 [Ythdc1-CKO]

data_processing:
The image data measured by the high-throughput sequencer are converted into  sequence data (reads) by CASAVA base recognition. Raw data (raw reads) of fastq  format were firstly processed through in-house perl scripts. In this step, clean data  (clean reads) were obtained by removing reads containing adapter, reads containing N  base and low quality reads from raw data. At the same time, Q20, Q30 and GC  content the clean data were calculated. All the downstream analyses were based on  the clean data with high quality.
Reference genome and gene model annotation files were downloaded from  genome website directly. Index of the reference genome was built using Hisat2  (v2.0.5) and paired-end clean reads were aligned to the mouse reference genome(GRCm38.p6) using Hisat2 (v2.0.5). We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.
featureCounts (v1.5.0-p3) was used to count the reads numbers mapped to each  gene. And then FPKM of each gene was calculated based on the length of the gene  and reads count mapped to this gene. FPKM, expected number of Fragments Per  Kilobase of transcript sequence per Millions base pairs sequenced, considers the  effect of sequencing depth and gene length for the reads count at the same time, and is  currently the most commonly used method for estimating gene expression levels.
Differential expression analysis of two  conditions/groups (two biological replicates per condition) was performed using the  DESeq2 R package (1.20.0). DESeq2 provide statistical routines for determining  differential expression in digital gene expression data using a model based on the  negative binomial distribution. The resulting P-values were adjusted using the  Benjamini and Hochberg’s approach for controlling the false discovery rate .  padj<=0.05 and |log2(foldchange)| >= 0 were set as the threshold for significantly  differential expression.
Assembly: mouse reference genome(GRCm38.p6)
Supplementary files format and content: Processed data files; excel; fpkm of each gene

extract_protocol:
Ythdc1flox/flox and Ythdc1-HKO mice were fed with normal chow for 9 weeks.Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany). Three independent biological replicates for each group were used for RNA-seq.
mRNA was purified from total RNA by using poly-T oligo-attached magnetic beads.  Fragmentation was carried out using divalent cations under elevated temperature in  First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using  random hexamer primer and M-MuLV Reverse Transcriptase, then use RNaseH to  degrade the RNA. Second strand cDNA synthesis was subsequently performed using  DNA Polymerase I and dNTP. Remaining overhangs were converted into blunt ends  via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA  fragments, Adaptor with hairpin loop structure were ligated to prepare for  hybridization. In order to select cDNA fragments of preferentially 370~420 bp in  length, the library fragments were purified with AMPure XP system (Beckman  Coulter, Beverly, USA). Then PCR amplification, the PCR product was purified by  AMPure XP beads, and the library was finally obtained.
After the library is qualified, the different libraries are pooling according to the  effective concentration and the target amount of data off the machine, then being  sequenced by the Illumina NovaSeq 6000. The end reading of 150bp pairing is  generated. The basic principle of sequencing is to synthesize and sequence at the same  time (Sequencing by Synthesis). Four fluorescent labeled dNTP, DNA polymerase  and splice primers were added to the sequenced flowcell and amplified. When the  sequence cluster extends the complementary chain, each dNTP labeled by  fluorescence can release the corresponding fluorescence. The sequencer captures the  fluorescence signal and converts the optical signal into the sequencing peak by  computer software, so as to obtain the sequence information of the fragment to be  tested.


title: [PRO-Seq] K562 cells, LP recombination, e1 only WT (no e2), rep2

description: Library name: Sample 12

value: LP recombination, e1 only WT (no e2)


title: [PRO-Seq] K562 cells, LP recombination, e1 only WT (no e2), rep1

description:
Library name: Sample 11
"merge" bw files are merged from rep1 and rep2 bw


title: H929 wildtype cells [HW]

city: Jiangsu Nanjing

first: Deng

middle: Zhen

last: Dong

address: Xianlin Roud 138

institute: Nanjing University of Chinese Medicine

description: HW

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm8 whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
Reads Per Kilobase of exon per Megabase of library size (FPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Data filtering steps.Prior to assembly, the low quality reads(1,reads containing sequencing adaptors; 2,reads containing sequencing primer;3, nucleotide with q quality score lower than 20 )were removed .software CutAdapter,parameters -o 5 -p 100
Read alignment software,software HISAT ,version 2.0 ,parameters -l fr-firststrand -mi 20 -mx 500000 -p 2 -b dta -q phred33-quals -x 9
perform expression level for mRNAs by calculating FPKM.stimate the expression levels of all transcripts.software StringTie ,version 1.3.0,parameters -b dta -q phred33-quals.
Genome_build: Beta_vulgaris.GCF_000511025.2_RefBeet-1.2.2
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample

source_name: Multiple Myeloma cells

extract_protocol:
Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number >7.0. Approximately 10 ug of total RNA representing a specific adipose type was subjected to isolate Poly (A) mRNA with poly-T oligoattached magnetic beads (Invitrogen). Following purification, the poly(A)- or poly(A)+ RNA fractions is fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with the protocol for the mRNA-Seq sample preparation kit (Illumina, San Diego, USA), the average insert size for the paired-end libraries was 300 bp (±50 bp). And then we performed the paired-end sequencing on an Illumina Novaseq™ 6000 at the (lc-bio, China) following the vendor's recommended protocol.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: genotype/variation

value: wildtype


title: H929 DEAF1-OE cells [HO]

description: HO


title: O2-Z7

description: Library name: O2-Z7


title: HKO_1 [Ythdc1-CKO]


title: ARP1 wildtype cells [AW]

description: AW


title: K562 cells, LP recombination, e1_mRUNX1-L, rep1

description:
Library name: Sample 17
"merge" bw files are merged from rep1 and rep2 bw

data_processing:
For all NGS sequencing data, the quality of FASTQ files was first accessed using FastQC, and the Illumina sequencing adaptors were trimmed using fastp. Note that one of the ATAC-seq samples was sequenced at 2 × 150 bp instead of 2 × 80 bp. To ensure consistency across samples, the raw FASTQ files for this sample were trimmed to 80 bp prior to any analysis.
To enable accurate alignment of sequencing reads to the eNMU locus in the recombinant cell lines, we built a custom genome for each recombinant TF motif mutant using the reform command line tool. Each custom genome incorporated the exact mutant sequence flanked by Bxb1 recombination sites. Alignment was performed using the bowtie2 aligner: for ATAC-seq, “-end-to-end --very-sensitive” mode was used, with a subsequent step to remove mitochondrial reads using samtools view; Picard MarkDuplicates was then used for deduplication.
To better normalize ATAC-seq datasets across different clonal lines, we applied a “reads-under-peaks” approach to calculate scaling factors for each sample. ATAC-seq peaks were called for each sample using HMMRATAC in the MACS3 software with “-u 90 -l 30 -c 10” parameters. To create a unified peak set for count normalization, we first identified reciprocal >50% overlaps between peaks from biological replicates using bedtools intersect "-f 0.50 -r". Consensus peak sets from each group of replicates were then merged to create a union peak set.
To generate the read count matrix for ATAC-seq datasets, read pairs overlapping union peaks were quantified using featureCounts. DESeq2 was then used to calculate scaling factors for normalization. bamCoverage was used to generate normalized bigwig files at a bin size of 1 bp.
Merged bigwig files from two biological replicates were generated using kentUtils of the UCSC Genome Browser.
Assembly: hg38
Supplementary files format and content: bigwig

extract_protocol:
ATAC-seq was performed on 50,000 K562 cells with all buffer compositions and reaction conditions following the published Omni-ATAC protocol (Corces et al., 2017) unless otherwise specified. Briefly, cells were pelleted, washed once with ice-cold PBS, resuspended in ice-cold Lysis Buffer, and incubated on ice for 3 min. Upon addition of Wash Buffer and gentle inversion, nuclei were pelleted, and supernatant (cytoplasm) was discarded. Nuclei were then resuspended gently in 50 μL transposition reaction mix containing 1 μL homemade Tn5 transposase and incubated at 37°C for 30 min with 1000 rpm shaking. DNA was purified using the MinElute PCR purification kit (Qiagen) and eluted in 21 μL volume.
The entire purified product (~20 μL) was mixed with 2.5 μL of each Nextera index primer (25 μM) and 25 μL NEBNext High-Fidelity 2X PCR Master Mix and subjected to a first round of PCR: initial extension 72°C for 5 min; initial denaturation 98°C 30s; 5 cycles of 98°C for 10 s, 63°C for 30 s, 72°C for 1 min. To determine additional cycles needed to avoid over-amplification, a qPCR analysis was performed using 5 μL of the first-round PCR reaction (Buenrostro et al., 2015). For all ATAC-seq libraries, 3~4 additional cycles of amplification were performed on the remaining 45 μL PCR reaction, followed by sequential cleanup using the MinElute PCR purification kit (Qiagen) and homebrew SPRI beads (1.5× bead ratio). DNA concentration was measured by the Qubit dsDNA High Sensitivity assay (Thermo Fisher). The libraries were pooled for sequencing on the Element Biosciences AVITI (2 × 80 bp paired-end sequencing) or Illumina NovaSeq X Plus platform (2 × 150 bp paired-end sequencing).

value: LP recombination, e1_mRUNX1-L


title: O2-Z5

description: Library name: O2-Z5


title: D1_2


title: ff_3 [Ythdc1flox/flox]


title: DEAF1-OE cells for RAD50 IP

data_processing:
Use the cutadapt program to remove the joint sequence in the original offline data
Use Trimmomatic program to remove low-quality sequences to get clean data
Use Fastqc program to count the amount of clean data, the ratio of q20 and q30
Use bowtie2 program to compare clean data to reference genome
Calculate the capture efficiency of the sequencing experiment and the coverage of the ChIP area and the average sequencing depth; use MACS2 to perform peak-calling on the genome; perform peak annotation; use MEME for motif detection of combined peaks; peak differences and annotations between samples.
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited text files include Peak annotation and related analysis for each Sample ...

extract_protocol:
add 37% formaldehyde to the culture medium with at least 107 cells(final concentration of formaldehyde is 1%), shake on a shaker for 10 minutes; stop formaldehyde fixation: add glycine solution to a final concentration of 0.125mol/L, pipette and mix;Wash the cells with PBS, scrape the cells, harvest the cells, and discard the supernatant as much as possible.Combine the antibody with protein A+G magnetic beads (Magna ChIP™ 16-663);Add lysate and protease inhibitor to the cells, and sonicate in ice water bath; take 20ul as Input sample and store at 4℃;Add the sample to the protein A+G magnetic beads bound to the antibody and mix overnight; wash the magnetic column to elute the fragments bound to the protein A+G magnetic beads, and Input samples were added to ProteinaseK together and de-crosslinked at 65℃ overnight; DNA was extracted with phenol chloroform and digested with RNase.
Repair DNA fragment ends, add A base at 3'end, connect sequencing adapter; PCR amplification and fragment size selection of DNA productsm;After the library is qualified, it is sequenced through the Illumina HiSeq/NextSeq platform.

value: overexpression

tag: chip antibody

value: Human bone marrow


title: ARP1 DEAF1-OE cells [AO]

description: AO


title: O1-G7

description: Library name: O1-G7

value: 0-10cm soil


title: ff_2 [Ythdc1flox/flox]


title: Schmidtea mediterranea, whole-body, hnf4(RNAi), Replicate 3

city: Exeter

first: Jordi

last: Solana

address: Northcote House

department: Health and Life Sciences

country: United Kingdom

institute: University of Exeter

description:
Library name: HNF4_R3
bulk RNA-Seq knock-down

data_processing:
Reads were mapped to the reference genome using kallisto (https://github.com/pachterlab/kallisto).
Assembly: Genome assembly from (https://doi.org/10.1101/2023.12.22.572568) will be available on planmine (https://planmine.mpibpc.mpg.de/planmine/genome.do)
Supplementary files format and content: raw 'counts'

molecule: polyA RNA

organism: Schmidtea mediterranea

source_name: whole-body

growth_protocol: An asexual clonal line of the planarian species Schmidtea mediterranea was maintained at 18-20ºC in 1x PAM water and fed once a week with organic veal liver and starved 7 days minimum before any experimental procedure.

extract_protocol: Total RNA was isolated using 500 μL of TRIzolTM Reagent and later purified with  100 μL of chloroform, 250 µL of 2-propanol, washed in 500 µL of 75% ethanol and resuspended in 20 µL of nuclease-free water.

treatment_protocol: Worms of 6mm in length were injected with the respective treatment of dsRNA in a final concetration of 1000 ng/µL

value: whole-body


title: ff_1 [Ythdc1flox/flox]


title: HKO_3 [Ythdc1-CKO]


title: Schmidtea mediterranea, whole-body, hnf4(RNAi), Replicate 2

description:
Library name: HNF4_R2
bulk RNA-Seq knock-down


title: 24 h biofilms grown in TSB, BR3

city: Oviedo

first: Lucia

last: Fernandez

address: C/ Francisco Pintado Fe 26

country: Spain

institute: Instituto de Productos Lacteos de Asturias (IPLA-CSIC)

description: Library name: Sample 6

data_processing:
Quality of the sequences was verified with FastQC 0.11.2
EDGE-pro 1.3.1 workflow was employed for read processing. The pipeline as described by T.Magoc, et al., Evolutionary Bioinformatics, 2013, employs Bowtie2 (v2.2.3) to map reads to the reference genome (NCTC 8325), filters multimapped reads based on scoring alignments and determines read coverage for each genomic position and gene.
EDGE-pro output files were formated using the edgeToDeseq script provided in the EDGE-pro package and used as input for DESeq2 (v1.12.4) to calculate RPKMs per gene and quantify differential expression between conditions.
Assembly: S. aureus NCTC 8325
Supplementary files format and content: test_DESeq2results_normalized.csv: plain text file with comma-separated values. The file includes RPKM values for NCTC8325 genes in milk and TSB replicates as well as differential expression estimations, expressed as a log2 fold change between conditions.

organism: Staphylococcus aureus

growth_protocol: Biofilms were grown in TSB supplemented with 0.25% glucose or milk for 24 hours at 37 ºC in a static incubator. The planktonic phase was removed and the adhered cells were washed and harvested for RNA purification.

extract_protocol:
Bacterial cells were harvested, treated with RNA protect (Qiagen) and kept at -80ºC until RNA purification. RNA was obtained by mechanical disruption in the presence of acid phenol and subsequent purification was carried out with illustra RNA spin minikit (GE Healthcare)
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: strain


title: O2-Z9

description: Library name: O2-Z9


title: 24 h biofilms grown in TSB, BR2

description: Library name: Sample 5


title: Schmidtea mediterranea, whole-body, hnf4(RNAi), Replicate 1

description:
Library name: HNF4_R1
bulk RNA-Seq knock-down


title: K562 cells, LP recombination, e1_mRUNX1-L, rep2

description: Library name: Sample 18


title: O1-G8

description: Library name: O1-G8


title: 24 h biofilms grown in TSB, BR1

description: Library name: Sample 4


title: Schmidtea mediterranea, whole-body, hnf4(RNAi)+nkx2-2(RNAi), Replicate 3

description:
Library name: HNF4_NKX_R3
bulk RNA-Seq knock-down


title: BMMSCs, em-eNMs treated, rep3

city: Beijing

first: Liyuan

address: 22th Zhongguancun South Ave

institute: Department of Orthodontics,  Peking University School and Hospital of Stomatology, Beijing

description: Library name: EM-eNMs3bmmsc

data_processing:
Samples were then sequenced on an Illumina HiSeq 3000 platform. Quality control of the RNA-seq data was performed using FastQC (v0.11.5) and the FASTX toolkit (0.0.13). The data were mapped to the human reference genome (NCBI build 37, Jul 2007) using HISAT2 (v.2.0.4). Three samples per group were used. Differentially expressed genes and transcripts were identified using Ballgown software (v.3.4.0). Genes were considered significantly differentially expressed if they showed a ≥1.5-fold change and a P value < 0.05.
Assembly: human reference genome (NCBI build 37, Jul 2007)
Supplementary files format and content: counts, FPKM values for each Sample

source_name: bone marrow

extract_protocol:
Total RNAs from aged BMMSCs (P10–12), with or without em-eNMs treatment, were extracted by Trizol reagent (Thermo Fisher Scientific, Cat. No. 15596026).
Sequencing libraries were generated using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer's recommendations.

value: bone marrow

value: aged BMMSCs

tag: developmental stage

value: em-eNMs treated


title: BMMSCs, em-eNMs treated, rep1

description: Library name: EM-eNMs1bmmsc


title: Schmidtea mediterranea, whole-body, hnf4(RNAi)+nkx2-2(RNAi), Replicate 2

description:
Library name: HNF4_NKX_R2
bulk RNA-Seq knock-down


title: 24 h biofilms grown in milk BR3

description: Library name: Sample 3


title: O1-G9

description: Library name: O1-G9

data_processing:
Amplicon sequence variant (ASV) tables were constructed based on the DADA2 pipeline with default parameters. Read adapters were removed from raw read sequences using cutadapt, and read quality was inspected using FastQC. Chimeric sequences were removed from merged reads, and taxonomy was then assigned using the DADA2 "assignTaxonomy " function.
Assembly: metagenome
Supplementary files format and content: table files include ASV count and taxa
Supplementary files format and content: fasta files contain ASV sequences

extract_protocol:
DNeasy PowerSoil Kit (Qiagen Laboratories, Inc.)
Sequencing was carried out using Amplicon sequencing on Illumina MiSeq 2 × 250-bp PE platform


title: BMMSCs, untreated, rep3

description: Library name: Con3bmmsc

value: untreated


title: 24 h biofilms grown in milk, BR2

description: Library name: Sample 2


title: ITS O1-G10

description: Library name: ITS O1-G10


title: Schmidtea mediterranea, whole-body, hnf4(RNAi)+nkx2-2(RNAi), Replicate 1

description:
Library name: HNF4_NKX_R1
bulk RNA-Seq knock-down


title: 24 h biofilms grown in milk, BR1

description: Library name: Sample 1


title: BMMSCs, untreated, rep2

description: Library name: Con2bmmsc


title: O2-Z8

description: Library name: O2-Z8


title: Schmidtea mediterranea, whole-body, hnf4(RNAi)+gfp(RNAi), Replicate 3

description:
Library name: HNF4_GFP_R3
bulk RNA-Seq knock-down


title: DEAF1-OE cells for RAD50 input


title: ITS O1-G11

description: Library name: ITS O1-G11


title: BMMSCs, untreated, rep1

description: Library name: Con1bmmsc


title: Schmidtea mediterranea, whole-body, hnf4(RNAi)+gfp(RNAi), Replicate 2

description:
Library name: HNF4_GFP_R2
bulk RNA-Seq knock-down


title: ITS O1-L10

description: Library name: ITS O1-L10


title: Schmidtea mediterranea, whole-body, hnf4(RNAi)+gfp(RNAi), Replicate 1

description:
Library name: HNF4_GFP_R1
bulk RNA-Seq knock-down


title: O1-L1

description: Library name: O1-L1


title: BMMSCs, em-eNMs treated, rep2

description: Library name: EM-eNMs2bmmsc


title: 11_ID-79-PAM2-ODN_RelA_2

city: Houston

first: Scott

middle: E

last: Evans

state: TX

address: 6565 MD Anderson Blvd, Unit 1059

department: Pulmonary Medicine

institute: University of Texas MD Anderson Cancer Center

description:
Library name: 11_ID-79-PAM2-ODN_RelA_2
Column name in processed file: PO-2_RelA_GRCh38/ Tag Count in given bp (32609780.0 Total, normalization factor = 0.31, effective total = 10000000)

data_processing:
The single-end sequencing reads generated by Illumina sequencing are mapped to the genome hg38 using the BWA algorithm (“bwa aln/samse”) with default settings. Alignment information for each read is stored in the BAM format.
Only reads that pass Illumina’s purity filter, align with no more than 2 mismatches, and map uniquely to the genome are used in the subsequent analysis. In addition, duplicate reads (“PCR duplicates”) are removed.
Determination of fragment density of aligned reads ("tags") were calculated per sample, and combined per treatment with HOMER's makeTagDirectory function using default settings.
Regions of enriched tags ("peaks") were calculated per treatment against a pooled input, and combined per TF with HOMER's findPeaks and mergePeaks function using default settings. These combined peaks were used to define a TFBS consensus of DNA-binding regions per TF. The TFBS consensus was annotated, and tag numbers were calculated and normalized for all samples with HOMER's annotatePeak function using default settings.
Assembly: hg38
Supplementary files format and content: A TXT file containing TFBS tag counts, normalized using each directory by the total number of tags such that each directory contains 10 million tags.

source_name: HBEC3KT, Pam2+ODN, 4h, Rep2

growth_protocol: Cell cultures were maintained in filter sterilized growth medium (GM) of keratinocyte serum-free media supplemented with human recombinant epidermal growth factor and bovine pituitary extract and 1 percent volume/volume (% v/v) of antibiotic-antimycotic (100X).

extract_protocol:
Cell were harvested after a 5–10-minute incubation with 1 mL of 0.25 % trypsin-EDTA, further neutralized with 4 mL of 2 % v/v fetal bovine serum in filter sterilized phosphate-buffered saline (PBS). Detached cells were centrifuged at 250 g for 5 minutes at room temperature (25 ºC) and the resulting cell pellet was reconstituted in 10 mL of GM. Cell counting was performed. A frozen pellet of 10,000,000 cells per sample was prepared and sent according to Active Motif for their end-to-end ChIP-seq service according to manufacturer's instructions.
Chromatin preparation, sonication, sample quality/integrity assessment, ChIP and library generation, QC of NGS library, and sequencing using Illumina platform were performed using proprietary standard protocols and analysis by Active Motif.

treatment_protocol: Cells were exposed to 10 mL (for 100mm dishes) of fresh GM with either 1% v/v PBS, 4 µM Pam2 only, 1 µM ODN only, or both 4 µM Pam2 and 1 µM ODN (Pam2+ODN) and followed up for the indicated timepoints per experiment

value: Lung; Bronchus

value: Epithelial cell


title: ITS O1-L2

description: Library name: ITS O1-L2


title: 10_ID-78-PAM2-ODN_RelA_1

description:
Library name: 10_ID-78-PAM2-ODN_RelA_1
Column name in processed file: PO-1_RelA_GRCh38/ Tag Count in given bp (33287985.0 Total, normalization factor = 0.30, effective total = 10000000)

source_name: HBEC3KT, Pam2+ODN, 4h, Rep1


title: 09_ID-73-ODN_RelA_3

description:
Library name: 09_ID-73-ODN_RelA_3
Column name in processed file: ODN-3_RelA_GRCh38/ Tag Count in given bp (31539986.0 Total, normalization factor = 0.32, effective total = 10000000)

source_name: HBEC3KT, ODN, 4h, Rep3

value: ODN


title: Schmidtea mediterranea, whole-body, hnf4(RNAi)+foxF-1(RNAi), Replicate 2

description:
Library name: HNF4_FOXF_R2
bulk RNA-Seq knock-down


title: Schmidtea mediterranea, whole-body, hnf4(RNAi)+foxF-1(RNAi), Replicate 3

description:
Library name: HNF4_FOXF_R3
bulk RNA-Seq knock-down


title: 08_ID-72-ODN_RelA_2

description:
Library name: 08_ID-72-ODN_RelA_2
Column name in processed file: ODN-2_RelA_GRCh38/ Tag Count in given bp (32944611.0 Total, normalization factor = 0.30, effective total = 10000000)

source_name: HBEC3KT, ODN, 4h, Rep2


title: Schmidtea mediterranea, whole-body, hnf4(RNAi)+foxF-1(RNAi), Replicate 1

description:
Library name: HNF4_FOXF_R1
bulk RNA-Seq knock-down


title: 07_ID-71-ODN_RelA_1

description:
Library name: 07_ID-71-ODN_RelA_1
Column name in processed file: ODN-1_RelA_GRCh38/ Tag Count in given bp (34141642.0 Total, normalization factor = 0.29, effective total = 10000000)

source_name: HBEC3KT, ODN, 4h, Rep1


title: ITS O1-L4

description: Library name: ITS O1-L4

source_name: 0-10cm soil


title: Schmidtea mediterranea, whole-body, gfp(RNAi), Replicate 3

description:
Library name: GFP_R3
bulk RNA-Seq knock-down

value: WT


title: ITS O1-L5G5

description: Library name: ITS O1-L5G5


title: Schmidtea mediterranea, whole-body, gfp(RNAi), Replicate 2

description:
Library name: GFP_R2
bulk RNA-Seq knock-down


title: PBL141

type: RNA

city: Badalona

first: Miriam

last: Verdú-Bou

state: Barcelona

address: Ctra de Can Ruti, Camí de les Escoles, s/n

institute: Josep Carreras Leukaemia Research Institute

description: miRNA expression data from FFPE samples of PBL.

hyb_protocol: RNA was processed to add a Polu(A) tail and labeled with biotin. Then, was hybridized to each array according to the manufacturer's instructions.

scan_protocol: The Arrays were then washed using ThermoFisher fluidics stations, and scanned using the Gene Chip Scanner 3000.

data_processing:
The array image was acquired using ThermoFihser GeneChip® Operating Software. CHP data was generated with Gene Expression Console. The algorithm used was RMA
affymetrix-algorithm-name = rma-bg,quant-norm.sketch=0.usepm=true.bioc=true,pm-only,med-polish
affymetrix-algorithm-version = 1.0

label: Biotin

source_name: Homo sapiens PBL

label_protocol: As per manufacturer (ThermoFisher), with Biotin

extract_protocol: Total RNA extracction from formalin-fixed paraffin-embedded samples using RecoverAll Total Nucleic Acid Isolation according to the manufacturer's instructions.

tag: sample

value: FFPE

tag: group

value: Plasmablastic lymphoma (PBL)


title: 05_ID-65-PAM2_RelA_2

description:
Library name: 05_ID-65-PAM2_RelA_2
Column name in processed file: Pam2-2_RelA_GRCh38/ Tag Count in given bp (33748232.0 Total, normalization factor = 0.30, effective total = 10000000)

source_name: HBEC3KT, Pam2, 4h, Rep2

tag: tissue


title: ITS O1-L11

description: Library name: ITS O1-L11


title: 04_ID-64-PAM2_RelA_1

description:
Library name: 04_ID-64-PAM2_RelA_1
Column name in processed file: Pam2-1_RelA_GRCh38/ Tag Count in given bp (31424503.0 Total, normalization factor = 0.32, effective total = 10000000)

source_name: HBEC3KT, Pam2, 4h, Rep1

tag: cell type


title: PBL132


title: ITS O1-L3

description: Library name: ITS O1-L3


title: 06_ID-66-PAM2_RelA_3

description:
Library name: 06_ID-66-PAM2_RelA_3
Column name in processed file: Pam2-3_RelA_GRCh38/ Tag Count in given bp (29499475.0 Total, normalization factor = 0.34, effective total = 10000000)

source_name: HBEC3KT, Pam2, 4h, Rep3

tag: treatment


title: 03_ID-59-PBS_RelA_3

description:
Library name: 03_ID-59-PBS_RelA_3
Column name in processed file: PBS-3_RelA_GRCh38/ Tag Count in given bp (32434744.0 Total, normalization factor = 0.31, effective total = 10000000)

source_name: HBEC3KT, PBS, 4h, Rep3

tag: cell line

value: PBS


title: Schmidtea mediterranea, whole-body, gfp(RNAi), Replicate 1

description:
Library name: GFP_R1
bulk RNA-Seq knock-down


title: ITS O1-L8Z8

description: Library name: ITS O1-L8Z8


title: ITS O1-L9

description: Library name: ITS O1-L9


title: 12_ID-80-PAM2-ODN_RelA_3

description:
Library name: 12_ID-80-PAM2-ODN_RelA_3
Column name in processed file: PO-3_RelA_GRCh38/ Tag Count in given bp (34203398.0 Total, normalization factor = 0.29, effective total = 10000000)

source_name: HBEC3KT, Pam2+ODN, 4h, Rep3


title: Schmidtea mediterranea, whole-body, unperturbed conditions, scRNA-seq, batch 3, library 14_3 deep

description:
SPLiT-Seq
smed_cisreg_scrna_consensus_identities.RDS

data_processing:
Barcode processing and adapter trimming were made using SPLiT-seq toolbox (https://github.com/RebekkaWegmann/splitseq_toolbox), CutAdapt  v2.8 (https://github.com/marcelm/cutadapt), pairfq makepairs (https://github.com/sestaton/Pairfq) and Picard v2.21.1-SNAPSHOT (https://github.com/broadinstitute/picard)
Genome mapping was made with STAR-2.7.3a (https://github.com/alexdobin/STAR/releases/tag/2.7.3a)
Assembly: Genome assembly from (https://doi.org/10.1101/2023.12.22.572568) will be available on planmine (https://planmine.mpibpc.mpg.de/planmine/genome.do)
Supplementary files format and content: Tab-separated values files and .RDS Seurat object files

extract_protocol:
We used ACME dissociation to dissociate whole animals into fixed and permeable cell suspensions. Dissociated cells were labelled individually using a SPLiT-seq protocol (we sorted singlets by FACS during SPLiT-seq, right before the cell lysis step).
We followed the SPLiT-seq library construction pipeline (Dynabeads cDNA purification, template switching, PCR amplification, size selection, tagmentation, indexed-PCR amplification and second size selection). Size selection was performed using Roche KAPA Pure Beads at 0.6X-0.8X ratios. Tagmentation was performed either using the Nextera XT DNA Library Preparation Kit or Nextera DNA Library Preparation Kit (Illumina), according to the manufacturer's instructions.

treatment_protocol: L11_3 and L11_4 libraries were made from knockdown samples. Knockdown treatment consisted in injecting worms with 0.1 μg of gfp or hnf4 dsRNA for 3 consecutive days. Samples were processed 9 days post-injection.


title: 02_ID-58-PBS_RelA_2

description:
Library name: 02_ID-58-PBS_RelA_2
Column name in processed file: PBS-2_RelA_GRCh38/ Tag Count in given bp (31936857.0 Total, normalization factor = 0.31, effective total = 10000000)

source_name: HBEC3KT, PBS, 4h, Rep2


title: Schmidtea mediterranea, whole-body, knock-down conditions, scRNA-seq, batch 2, library 11_4 deep

description:
SPLiT-Seq
smed_cisreg_scrna_consensus_identities.RDS
smed_hnf_scrna.RDS
gfpi_hnf_cell_barcodes.tsv

value: gfpi and hnf4i


title: ITS O1-G2

description: Library name: ITS O1-G2


title: 01_ID-57-PBS_RelA_1

description:
Library name: 01_ID-57-PBS_RelA_1
Column name in processed file: PBS-1_RelA_GRCh38/ Tag Count in given bp (34010942.0 Total, normalization factor = 0.29, effective total = 10000000)

source_name: HBEC3KT, PBS, 4h, Rep1


title: ITS O1-L7

description: Library name: ITS O1-L7


title: Schmidtea mediterranea, whole-body, knock-down conditions, scRNA-seq, batch 2, library 11_3 deep


title: ITS O1-L6

description: Library name: ITS O1-L6


title: PBL140


title: 00_Pooled_Input_0

description:
Library name: 00_Pooled_Input_0
Column name in processed file: Input_RelA_GRCh38/ Tag Count in given bp (51429876.0 Total, normalization factor = 0.19, effective total = 10000000)

source_name: HBEC3KT, PBS, 4h, Input

value: Input


title: ITS O1-Z10

description: Library name: ITS O1-Z10


title: ITS O1-Z11

description: Library name: ITS O1-Z11


title: Schmidtea mediterranea, whole-body, unperturbed conditions, scRNA-seq, batch 1, library 8_4 deep

source_name: whole-body


title: PBL130


title: PBL138


title: PBL121


title: PBL114


title: PBL117


title: Schmidtea mediterranea, whole-body, unperturbed conditions, scRNA-seq, batch 3, library 14_4 deep


title: Schmidtea mediterranea, whole-body, unperturbed conditions, scATAC-seq, batch 1

data_processing:
Alignment: reads were aligned against reference genome using Bowtie2 software.
Filtering duplicates: duplicated pairs were removed using Samtools software.
Localization of the cleavage site: the enzyme cleavage site was determined as the position -4 (minus strand) or +5 (plus strand) from each read start, and this position was extended 5 bp in both directions.
Visualization: bigWig files were visualized using IGV Browser
Assembly: Genome assembly from (https://doi.org/10.1101/2023.12.22.572568) will be available on planmine (https://planmine.mpibpc.mpg.de/planmine/genome.do)
Supplementary files format and content: Tab-separated values files and .RDS Seurat object files

extract_protocol:
Animals were flash-frozen in liquid nitrogen and resuspended in cold lysis buffer. Animals were dissociated by smashing on a cell strainer with the aid of a syringe plunger. The resulting nuclei suspension was resuspended in 0.04% BSA, and later resuspended in tagmentation buffer and custom-loaded Tn5 transposase.
Tagmented DNA was purified using the Monarch PCR DNA Cleanup kit following manufacturer's instructions, and amplified using PCR with Kapa HiFi HotStart ReadyMix (Roche). The library was sequenced in a NovaSeq X Plus PE150 Illumina sequencer.


title: ITS O1-Z1G1

description: Library name: ITS O1-Z1G1


title: Schmidtea mediterranea, whole-body, unperturbed conditions, scRNA-seq, batch 1, library 8_3 deep

description:
SPLiT-Seq
smed_cisreg_scRNA_consensus_identities.RDS


title: 19_ID-53-PAM2ODN-4

description: Library name: 19_ID-53-PAM2ODN-4

data_processing:
The paired-end sequencing reads generated by Illumina sequencing are mapped to the genome hg38 using the STAR algorithm 2.5.2b with default settings. Alignment information for each read is stored in the BAM format.
Only read pairs that have both ends aligned are counted. Read pairs that have their two ends mapping to different chromosomes or mapping to same chromosome but on different strands are discarded.
Gene annotations we were obtained from Subread package. Annotations from NCBI RefSeq database were adapted by merging overlapping exons from the same gene to form a set of disjoint exons for each gene. Genes with the same Entrez gene identifiers were also merged into one gene.
Assembly: hg38
Supplementary files format and content: A XSLX file containing gene level counts, normalized using the FPKM (Fragments Per Kilobase of transcript per Million mapped reads) method.

source_name: HBEC3KT, Pam2+ODN, 4h, Rep4

extract_protocol:
Cell were harvested after a 5–10-minute incubation with 1 mL of 0.25 % trypsin-EDTA, further neutralized with 4 mL of 2 % v/v fetal bovine serum in filter sterilized phosphate-buffered saline (PBS). Detached cells were centrifuged at 250 g for 5 minutes at room temperature (25 ºC) and the resulting cell pellet was reconstituted in 10 mL of GM. Cell counting was performed using a 200 µL sample in a Scepter 3.0 handheld automated cell counter (60 µm sensors). A frozen pellet of 1,000,000 cells per sample was prepared and sent according to Active Motif for their end-to-end RNA-seq service according to manufacturer's instructions.
Isolation of total RNA, sample quality/integrity assessment, PolyA mRNA enrichment, directional library preparation (stranded method), QC of NGS library, and sequencing using Illumina platform were performed using proprietary standard protocols and analysis by Active Motif.


title: 18_ID-47-ODN-5

description: Library name: 18_ID-47-ODN-5

source_name: HBEC3KT, ODN, 4h, Rep5


title: PBL118


title: K562 cells, LP recombination, eNMU mutant library, FlowFISH unsorted, rep2

description:
Library name: Sample 18
Column name in barcode_counts_across_bins.xlsx: BioRep2, Unsorted

data_processing:
For all NGS sequencing data, the quality of FASTQ files was first accessed using FastQC, and the Illumina sequencing adaptors were trimmed using fastp.
The 8-bp element barcodes were extracted from the trimmed FASTQ Read 1 using fastx_trimmer with flags -Q33 -f 23 -l 30. The FASTQ format was converted into FASTA format using seqtk with the command “seqtk seq -a -q20 -n N”. Occurrences of each barcode were counted using fastx_collapser. The 328 unique barcodes were associated with their corresponding 83 elements using a custom python script. Processed barcode read counts across bins and calculated barcode activity scores are provided in Table S3 of the manuscript
Supplementary files format and content: barcode_counts_across_bins.xlsx

extract_protocol:
To measure enhancer activity of individual elements within the pooled recombinant population, HCR-FlowFISH was performed following the published protocol (Reilly et al., 2021) precisely, including all solution volumes and centrifugation parameters, prior to cell sorting. Briefly, 20 million recombinant cells of each biological replicate were fixed with 4% formaldehyde in PBST (1× PBS, 0.1% Tween 20) at room temperature for 1 h and washed with PBST for 4 times. Following 10 min up to 24 h incubation with cold 70% Ethanol at 4°C, cells were washed with PBST twice and incubated with the pre-warmed Probe Hybridization Buffer at 37°C for 30 min. HCR probes for NMU and ACTB were added together to cells to reach a final concentration of 4 nM per probe. The samples were then incubated overnight with agitation in a 37°C hybridization oven. On the next day, cells were washed with the Probe Wash Buffer for 5 times, with 5× SSCT (5× SSC, 0.1% Tween 20) once, and pre-amplified in the Amplification Buffer for 30 min at room temperature. Snap-cooled hairpins were diluted in the Amplification Buffer and then added to the pre-amplified samples to reach a final hairpin concentration of 60 nM. Samples were incubated with rotation in a dark room overnight at room temperature. On the next day, 5× volume of 5× SSCT was added to the samples before centrifugation and removal of the hairpin amplification solution. Cells were then washed with 5× SSCT for 6 times before final resuspension in PBS at a density of 10 million cells/mL. The samples were filtered through a 35 µm Cell Strainer cap into a 5 mL polystyrene tube (Corning) before sorting. Cells were sorted into 8 bins (2 rounds of 4-way sorting) based on the AF647/AF488 ratio at a minimum coverage of 500× barcode coverage per bin to ensure robust representation in the sequencing library. Sorted cells, together with the unsorted background sample, were pelleted and resuspended in 400 µL of ChIP lysis buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, 1% SDS), and de-crosslinked overnight at 65°C with 1000× rpm shaking. Samples were then treated with RNase A (Thermo Scientific) and Proteinase K (Invitrogen) before phenol-chloroform extraction of genomic DNA (gDNA).
Sequencing libraries were prepared by two rounds of PCR using Q5 High-Fidelity 2X Master Mix (NEB). The 1st round PCR was performed on the recovered gDNA corresponding to a minimum of 200× barcode coverage, using primers ZZ145 and ZZ146 (Table S2 of manuscript) to specifically amplify the 8-bp barcodes from the eNMU genomic locus. A maximum of 500 ng gDNA was used as input in a 50 µL PCR reaction. The PCR program was: initial denaturation 98 °C for 3 min; 11 cycles of 98°C for 10 s, 65°C for 30 s, 72°C for 1 min; and final extension 72°C for 5 min. The PCR products were then purified using homebrew SPRI beads (1.5× bead ratio) to remove unused primers, followed by the 2nd round PCR with standard Illumina Nextera primers to append sequencing library indices and flow cell adaptors to the amplicons. The PCR program was: initial denaturation 98°C for 30 s; 11 cycles of 98°C for 10 s, 67°C for 30 s, 72°C for 20 s; and final extension 72°C for 5 min. Final PCR products were purified using the MinElute PCR purification kit (Qiagen) and DNA concentration was measured by the Qubit dsDNA High Sensitivity assay (Thermo Fisher). The libraries were pooled for sequencing on the Element Biosciences AVITI platform (2 × 80 bp paired-end sequencing).

value: erythroleukemia

value: LP recombination, eNMU mutant libray in bulk


title: ITS O1-Z2

description: Library name: ITS O1-Z2

value: 0-10cm soil


title: ITS O1-Z3

description: Library name: ITS O1-Z3


title: 17_ID-46-ODN-4

description: Library name: 17_ID-46-ODN-4

source_name: HBEC3KT, ODN, 4h, Rep4


title: K562 cells, LP recombination, eNMU mutant library, FlowFISH Bin8, rep2

description:
Library name: Sample 17
Column name in barcode_counts_across_bins.xlsx: BioRep2, Bin8


title: 20_ID-54-PAM2ODN-5

description: Library name: 20_ID-54-PAM2ODN-5

source_name: HBEC3KT, Pam2+ODN, 4h, Rep5


title: PBL106


title: PBL122


title: PBL110


title: PBL116


title: 16_ID-41-PAM2-5

description: Library name: 16_ID-41-PAM2-5

source_name: HBEC3KT, Pam2, 4h, Rep5


title: K562 cells, LP recombination, eNMU mutant library, FlowFISH Bin7, rep2

description:
Library name: Sample 16
Column name in barcode_counts_across_bins.xlsx: BioRep2, Bin7


title: ITS O1-Z5

description: Library name: ITS O1-Z5


title: PBL111


title: PBL112


title: PBL105


title: ITS O1-Z6

description: Library name: ITS O1-Z6


title: 14_ID-34-PBS-5

description: Library name: 14_ID-34-PBS-5

source_name: HBEC3KT, PBS, 4h, Rep5


title: 15_ID-40-PAM2-4

description: Library name: 15_ID-40-PAM2-4

source_name: HBEC3KT, Pam2, 4h, Rep4


title: PBL102

source_name: Homo sapiens PBL


title: K562 cells, LP recombination, eNMU mutant library, FlowFISH Bin5, rep2

description:
Library name: Sample 14
Column name in barcode_counts_across_bins.xlsx: BioRep2, Bin5


title: ITS O1-Z7

description: Library name: ITS O1-Z7

source_name: 0-10cm soil


title: 13_ID-33-PBS-4

description: Library name: 13_ID-33-PBS-4

source_name: HBEC3KT, PBS, 4h, Rep4


title: K562 cells, LP recombination, eNMU mutant library, FlowFISH Bin4, rep2

description:
Library name: Sample 13
Column name in barcode_counts_across_bins.xlsx: BioRep2, Bin4


title: ITS O1-G3

description: Library name: ITS O1-G3


title: K562 cells, LP recombination, eNMU mutant library, FlowFISH Bin3, rep2

description:
Library name: Sample 12
Column name in barcode_counts_across_bins.xlsx: BioRep2, Bin3


title: ITS O1-Z9

description: Library name: ITS O1-Z9


title: ITS O1-Z4

description: Library name: ITS O1-Z4


title: PBL100

description: miRNA expression data from FFPE samples of PBL.


title: PBL099


title: 11_ID-39-PAM2ODN-2

description: Library name: 11_ID-39-PAM2ODN-2

source_name: HBEC3KT, Pam2+ODN, 4h, Rep2


title: K562 cells, LP recombination, eNMU mutant library, FlowFISH Bin2, rep2

description:
Library name: Sample 11
Column name in barcode_counts_across_bins.xlsx: BioRep2, Bin2


title: 10_ID-50-PAM2ODN-1

description: Library name: 10_ID-50-PAM2ODN-1

source_name: HBEC3KT, Pam2+ODN, 4h, Rep1


title: PBL095


title: ITS O2-G1

description: Library name: ITS O2-G1


title: K562 cells, LP recombination, eNMU mutant library, FlowFISH Bin6, rep2

description:
Library name: Sample 15
Column name in barcode_counts_across_bins.xlsx: BioRep2, Bin6


title: K562 cells, LP recombination, eNMU mutant library, FlowFISH Bin1, rep2

description:
Library name: Sample 10
Column name in barcode_counts_across_bins.xlsx: BioRep2, Bin1


title: ITS O2-G10

description: Library name: ITS O2-G10


title: K562 cells, LP recombination, eNMU mutant library, FlowFISH unsorted, rep1

description:
Library name: Sample 9
Column name in barcode_counts_across_bins.xlsx: BioRep1, Unsorted


title: PBL094


title: 09_ID-45-ODN-3

description: Library name: 09_ID-45-ODN-3

source_name: HBEC3KT, ODN, 4h, Rep3


title: 08_ID-44-ODN-2

description: Library name: 08_ID-44-ODN-2

source_name: HBEC3KT, ODN, 4h, Rep2


title: 12_ID-40-PAM2ODN-3

description: Library name: 12_ID-40-PAM2ODN-3

source_name: HBEC3KT, Pam2+ODN, 4h, Rep3


title: 07_ID-43-ODN-1

description: Library name: 07_ID-43-ODN-1

source_name: HBEC3KT, ODN, 4h, Rep1


title: ITS O2-G3

description: Library name: ITS O2-G3


title: ITS O2-G11

description: Library name: ITS O2-G11


title: PBL092


title: K562 cells, LP recombination, eNMU mutant library, FlowFISH Bin8, rep1

description:
Library name: Sample 8
Column name in barcode_counts_across_bins.xlsx: BioRep1, Bin8


title: PBL097


title: ITS O2-G2

description: Library name: ITS O2-G2


title: PBL093


title: K562 cells, LP recombination, eNMU mutant library, FlowFISH Bin7, rep1

description:
Library name: Sample 7
Column name in barcode_counts_across_bins.xlsx: BioRep1, Bin7


title: K562 cells, LP recombination, eNMU mutant library, FlowFISH Bin6, rep1

description:
Library name: Sample 6
Column name in barcode_counts_across_bins.xlsx: BioRep1, Bin6


title: ITS O2-G4

description: Library name: ITS O2-G4


title: 05_ID-37-PAM2-2

description: Library name: 05_ID-37-PAM2-2

source_name: HBEC3KT, Pam2, 4h, Rep2


title: K562 cells, LP recombination, eNMU mutant library, FlowFISH Bin5, rep1

description:
Library name: Sample 5
Column name in barcode_counts_across_bins.xlsx: BioRep1, Bin5


title: PBL090


title: 04_ID-39-PAM2-1

description: Library name: 04_ID-39-PAM2-1

source_name: HBEC3KT, Pam2, 4h, Rep1


title: K562 cells, LP recombination, eNMU mutant library, FlowFISH Bin4, rep1

description:
Library name: Sample 4
Column name in barcode_counts_across_bins.xlsx: BioRep1, Bin4


title: ITS O2-G6

description: Library name: ITS O2-G6


title: PBL089


title: PBL087


title: ITS O2-G7

description: Library name: ITS O2-G7


title: PBL085 borderline to DLBCL


title: 03_ID-32-PBS-3

description: Library name: 03_ID-32-PBS-3

source_name: HBEC3KT, PBS, 4h, Rep3


title: PBL081


title: K562 cells, LP recombination, eNMU mutant library, FlowFISH Bin3, rep1

description:
Library name: Sample 3
Column name in barcode_counts_across_bins.xlsx: BioRep1, Bin3


title: PBL084 borderline to DLBCL


title: K562 cells, LP recombination, eNMU mutant library, FlowFISH Bin2, rep1

description:
Library name: Sample 2
Column name in barcode_counts_across_bins.xlsx: BioRep1, Bin2


title: 06_ID-38-PAM2-3

description: Library name: 06_ID-38-PAM2-3

source_name: HBEC3KT, Pam2, 4h, Rep3


title: ITS O2-G8

description: Library name: ITS O2-G8


title: ITS O2-G5

description: Library name: ITS O2-G5


title: ITS O1-G4

description: Library name: ITS O1-G4


title: ITS O2-L1

description: Library name: ITS O2-L1


title: K562 cells, LP recombination, eNMU mutant library, FlowFISH Bin1, rep1

description:
Library name: Sample 1
Column name in barcode_counts_across_bins.xlsx: BioRep1, Bin1


title: 01_ID-29-PBS-1

description: Library name: 01_ID-29-PBS-1

source_name: HBEC3KT, PBS, 4h, Rep1


title: [PRO-Seq] K562 cells, LP recombination, e1_mRARA, rep1

description:
Library name: Sample 29
"merge" bw files are merged from rep1 and rep2 bw

value: LP recombination, e1_mRARA


title: HBEC3KT, Pam2+ODN, 4h, Rep2

description:
Library name: 11_ID-79-PAM2-ODN_cJun_2
Column name in processed file: PO-2_cJun_GRCh38/ Tag Count in given bp (50793920.0 Total, normalization factor = 0.20, effective total = 10000000)

source_name: Lung; Bronchus


title: [PRO-Seq] K562 cells, LP recombination, LTRen_mKLF, rep2

description: Library name: Sample 28

value: LP recombination, LTRen_mKLF


title: [PRO-Seq] K562 cells, LP recombination, e1_mRARA, rep2

description: Library name: Sample 30


title: ITS O2-G9Z10

description: Library name: ITS O2-G9Z10


title: ITS O2-L10

description: Library name: ITS O2-L10


title: HBEC3KT, Pam2+ODN, 4h, Rep1

description:
Library name: 10_ID-78-PAM2-ODN_cJun_1
Column name in processed file: PO-1_cJun_GRCh38/ Tag Count in given bp (46230396.0 Total, normalization factor = 0.22, effective total = 10000000)


title: PBL074 borderline to plasmacytoma


title: PBL078 borderline to plasmacytoma


title: HBEC3KT, ODN, 4h, Rep3

description:
Library name: 09_ID-73-ODN_cJun_3
Column name in processed file: ODN-3_cJun_GRCh38/ Tag Count in given bp (44871866.0 Total, normalization factor = 0.22, effective total = 10000000)


title: PBL075

description: miRNA expression data from FFPE samples of PBL.


title: PBL073 unconventional clinic


title: PBL076


title: PBL071


title: ITS O2-L11

description: Library name: ITS O2-L11


title: HBEC3KT, ODN, 4h, Rep2

description:
Library name: 08_ID-72-ODN_cJun_2
Column name in processed file: ODN-2_cJun_GRCh38/ Tag Count in given bp (37221932.0 Total, normalization factor = 0.27, effective total = 10000000)


title: HBEC3KT, ODN, 4h, Rep1

description:
Library name: 07_ID-71-ODN_cJun_1
Column name in processed file: ODN-1_cJun_GRCh38/ Tag Count in given bp (37205961.0 Total, normalization factor = 0.27, effective total = 10000000)


title: ITS O2-L3

description: Library name: ITS O2-L3


title: [PRO-Seq] K562 cells, LP recombination, LTRen_mKLF, rep1

description:
Library name: Sample 27
"merge" bw files are merged from rep1 and rep2 bw


title: ITS O2-L2

description: Library name: ITS O2-L2


title: [PRO-Seq] K562 cells, LP recombination, LTRpr_mKLF, rep2

description: Library name: Sample 26

value: LP recombination, LTRpr_mKLF


title: HBEC3KT, Pam2, 4h, Rep3

description:
Library name: 06_ID-66-PAM2_cJun_3
Column name in processed file: Pam2-3_cJun_GRCh38/ Tag Count in given bp (36924005.0 Total, normalization factor = 0.27, effective total = 10000000)


title: [PRO-Seq] K562 cells, LP recombination, LTRpr_mKLF, rep1

description:
Library name: Sample 25
"merge" bw files are merged from rep1 and rep2 bw


title: HBEC3KT, Pam2, 4h, Rep2

description:
Library name: 05_ID-65-PAM2_cJun_2
Column name in processed file: Pam2-2_cJun_GRCh38/ Tag Count in given bp (37485947.0 Total, normalization factor = 0.27, effective total = 10000000)


title: ITS O2-L4

description: Library name: ITS O2-L4


title: ITS O2-L5Z6

description: Library name: ITS O2-L5Z6


title: [PRO-Seq] K562 cells, LP recombination, e1_mRUNX1-L, rep2

description: Library name: Sample 24

value: LP recombination, e1_mRUNX1-L


title: [PRO-Seq] K562 cells, LP recombination, e1_mRUNX1-L, rep1

description:
Library name: Sample 23
"merge" bw files are merged from rep1 and rep2 bw


title: HBEC3KT, Pam2, 4h, Rep1

description:
Library name: 04_ID-64-PAM2_cJun_1
Column name in processed file: Pam2-1_cJun_GRCh38/ Tag Count in given bp (42068924.0 Total, normalization factor = 0.24, effective total = 10000000)


title: HBEC3KT, PBS, 4h, Rep3

description:
Library name: 03_ID-59-PBS_cJun_3
Column name in processed file: PBS-3_cJun_GRCh38/ Tag Count in given bp (35471223.0 Total, normalization factor = 0.28, effective total = 10000000)


title: PBL069 unconventional clinic


title: ITS O2-L6

description: Library name: ITS O2-L6


title: ITS O2-L7

description: Library name: ITS O2-L7


title: [PRO-Seq] K562 cells, LP recombination, e2_mSTAT5, rep2

description: Library name: Sample 22

value: LP recombination, e2_mSTAT5


title: 02_ID-30-PBS-2

description: Library name: 02_ID-30-PBS-2

source_name: HBEC3KT, PBS, 4h, Rep2

value: Lung; Bronchus


title: HBEC3KT, PBS, 4h, Rep2

description:
Library name: 02_ID-58-PBS_cJun_2
Column name in processed file: PBS-2_cJun_GRCh38/ Tag Count in given bp (36550297.0 Total, normalization factor = 0.27, effective total = 10000000)

value: Epithelial cell


title: PBL061borderline plasmacytoma


title: PBL063 unconventional clinic


title: PBL060 unconventional clinic


title: PBL068 borderline to DLBCL


title: [PRO-Seq] K562 cells, LP recombination, e2_mSTAT5, rep1

description:
Library name: Sample 21
"merge" bw files are merged from rep1 and rep2 bw


title: [PRO-Seq] K562 cells, LP recombination, e1_mRUNX1, rep2

description: Library name: Sample 20

value: LP recombination, e1_mRUNX1


title: PBL059


title: PBL064 unconventional clinic


title: ITS O1-G6

description: Library name: ITS O1-G6


title: LIB5455520_SAM24414654_S6_L001

description:
SAM24414654_LIB5455520
NextSeq1

source_name: Colon

value: Colon

value: CRC line

tag: molecule type

value: CMO


title: ITS O2-L8

description: Library name: ITS O2-L8


title: HBEC3KT, PBS, 4h, Rep1

description:
Library name: 01_ID-57-PBS_cJun_1
Column name in processed file: PBS-1_cJun_GRCh38/ Tag Count in given bp (46147573.0 Total, normalization factor = 0.22, effective total = 10000000)

value: PBS


title: [PRO-Seq] K562 cells, LP recombination, e1_mRUNX1, rep1

description:
Library name: Sample 19
"merge" bw files are merged from rep1 and rep2 bw

tag: genotype


title: [PRO-Seq] K562 cells, LP recombination, e1_mGATA1, rep2

description: Library name: Sample 18

value: LP recombination, e1_mGATA1


title: LIB5455519_SAM24414654_S2_L001

description:
SAM24414654_LIB5455519
NextSeq1


title: ITS O2-L9

description: Library name: ITS O2-L9


title: ITS O2-Z1

description: Library name: ITS O2-Z1


title: LIB5455523_SAM24414655_S3_L001

description:
SAM24414655_LIB5455523
NextSeq1


title: ITS O2-Z11

description: Library name: ITS O2-Z11


title: [PRO-Seq] K562 cells, LP recombination, e1_mGATA1, rep1

description:
Library name: Sample 17
"merge" bw files are merged from rep1 and rep2 bw


title: ITS O2-Z2

description: Library name: ITS O2-Z2


title: [PRO-Seq] K562 cells, LP recombination, WT_eNMU, rep1

description:
Library name: Sample 15
"merge" bw files are merged from rep1 and rep2 bw

value: LP recombination, WT_eNMU


title: PBL056

source_name: Homo sapiens PBL

value: FFPE

value: Plasmablastic lymphoma (PBL)


title: LIB5455522_SAM24414655_S5_L001

description:
SAM24414655_LIB5455522
NextSeq1


title: ITS O2-Z3

description: Library name: ITS O2-Z3

source_name: 0-10cm soil

value: 0-10cm soil


title: K562 cells, LP recombination, WT_eNMU, rep2 (Input)

description: Library name: Sample 10

data_processing:
For all NGS sequencing data, the quality of FASTQ files was first accessed using FastQC, and the Illumina sequencing adaptors were trimmed using fastp.
To enable accurate alignment of sequencing reads to the eNMU locus in the recombinant cell lines, we built a custom genome for each recombinant TF motif mutant using the reform command line tool. Each custom genome incorporated the exact mutant sequence flanked by Bxb1 recombination sites. Alignment was performed using the bowtie2 aligner: for ChIP-seq, “-end-to-end --very-sensitive” mode was used; Picard MarkDuplicates was then used for deduplication.
To better normalize genomics datasets across different clonal lines, we applied a “reads-under-peaks” approach to calculate scaling factors for each sample. ChIP-seq peaks were called for each biological replicate and for pooled replicates using MACS2 with the input samples as control and the parameters “--broad --broad-cutoff 0.05 --keep-dup all”. Consensus peak sets from each group of replicates was generated using a published bash script (Additional file 5 of Reske et al., 2020), which identifies pooled peaks that show >50% reciprocal overlap with each biological replicate. These consensus peak sets were then merged to create a union peak set.
To generate the read count matrix for ChIP-seq datasets, read pairs overlapping union peaks were quantified using featureCounts. DESeq2 was then used to calculate scaling factors for normalization. bamCoverage was used to generate normalized bigwig files at a bin size of 50 bp for ChIP-seq.
Merged bigwig files from two biological replicates were generated using kentUtils of the UCSC Genome Browser.
Assembly: hg38
Supplementary files format and content: bigwig

extract_protocol:
Cells were first crosslinked with 2 mM disuccinimidyl glutarate (Santa Cruz) at room temperature for 30 min, washed 3 times with PBS and then crosslinked with 1% formaldehyde at room temperature for 5 min before quenching with 200 mM glycine at room temperature for 5 min. Two additional PBS washes were performed, and cell pellets were lysed with Farnham Lysis Buffer (5 mM PIPES, pH 8, 85 mM KCl, 0.5% NP40, 10 mM glycine, 1× Thermo Scientific Pierce Protease Inhibitor) on ice for 20 min. After centrifugation and supernatant removal, the nuclear pellet was resuspended in RIPA Lysis Buffer (10 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1× Thermo Scientific Pierce Protease Inhibitor) and incubated on ice for 10 min. Sonication was carried out using a Diagenode Bioruptor device at High Setting, 30 sec on/30 sec off for three rounds of 10-min cycle to shear chromatin to a size of 100~300 bp. The lysate was then clarified by centrifugation at 20,000 r.c.f., 4°C for 15 min, of which 2% was kept as ChIP input. Each IP used 4 million cells/4 µg antibody/40 µL Dynabeads Protein G (Thermo Scientific). Beads were washed three times with 5 mg/mL BSA in PBS and incubated with corresponding antibodies at 4°C for 6 h to overnight with rotation. Another three BSA/PBS washes were performed to remove unbound antibodies, and the clarified chromatin lysate was added to the beads and incubated overnight at 4°C with rotation. Beads were then washed with the following buffers, each for three times: Low Salt Wash Buffer (20 mM Tri-HCl, pH 8, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% SDS), High Salt Wash Buffer (20 mM Tri-HCl, pH 8, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS), and LiCl Wash Buffer (10 mM Tri-HCl, pH 8, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate). After one final wash with TE Buffer (10 mM Tris-Cl, pH 8, 1 mM EDTA), chromatin was eluted from beads by two rounds of incubation with ChIP elution buffer (1% SDS, 0.1 M sodium bicarbonate). Each incubation involved 15 min shaking at 1200 rpm at 65°C, followed by 15 min rotation at room temperature. The eluates and input samples were treated with RNase A at 37°C for 30 min, de-crosslinked at 65°C overnight with 900 rpm shaking, followed by Proteinase K treatment at 45°C for 2 h with shaking. DNA was purified using MinElute PCR Purification Kit (Qiagen).
For p300 ChIP, sequencing libraries were prepared using a Tn5 tagmentation-based protocol. Briefly, 20 μL tagmentation reactions were set up with 0.35 ng of ChIP DNA or 1 ng of input DNA, 1× TAPS-DMF Buffer (10 mM TAPS-NaOH, pH 8.5, 5 mM MgCl2, 10% DMF), and 1 μL of 1:15 diluted homemade Tn5 transposase (a kind gift from Dr. Roman Spektor). The reaction was incubated at 55°C for 10 min, and 2 μL of 1% SDS was added immediately, followed by another 55°C incubation for 7 min to strip off Tn5 from DNA. Post-tagmentation PCR was carried out in 100 μL volume with 10 μL of the tagmentation reaction, 1× Phusion HF Buffer, 200 μM dNTPs, 400 nM of each Illumina Nextera index primer, and 1 μL of homemade Phusion polymerase. The PCR program was: initial extension 72°C for 3 min; initial denaturation 98°C 30s; 13 cycles of 98°C for 10 s, 63°C for 30 s, 72°C for 3 min; and final extension 72°C for 5 min. PCR products were purified first using the MinElute PCR purification kit (Qiagen), followed by an additional cleanup with homemade SPRI beads (1.5× bead ratio) to ensure complete removal of unused primers. DNA concentration was measured by the Qubit dsDNA High Sensitivity assay (Thermo Fisher). The libraries were pooled for sequencing on the Element Biosciences AVITI platform (2 × 80 bp paired-end sequencing).

value: erythroleukemia


title: LIB5455521_SAM24414655_S4_L001

description:
SAM24414655_LIB5455521
NextSeq1

data_processing:
CMO data was processed using CellRanger and scRNA-Seq was processed using STARsolo with standard parameters.
Cumulus workflow with default settings was used for demultiplexing
Assembly: GRCh38 (GENCODE v32/Ensembl 98)

extract_protocol:
Cells from each sample were washed and re-suspended in 180µl of PBS+1%BSA and transferred to a round-bottom 96-well plate. Then 20µl of each anchor-barcode mix was added to the suspension for a final concentration of 2µM. After the incubation for 15 minutes at room temperature, 20µl of co-anchor was added to each sample (final concentration of 2µM) and incubate for another 15 minutes at RT. Cells were then washed by 500µl of cold PBS+1% BSA twice before pooling together.
The library construction was done following the manufacturer’s manual with a few modifications to accommodate the MULTI-seq protocol: (1) during the cDNA amplification 1µl of the following primer (10µM) was added to the master mix, 5’-CTTGGCACCCGAGAATTCC-3’; (2) the supernatant from the 0.6X SPRI beads cleanup for cDNA was kept and further cleaned up by using 2.0X SPRI beads; (3) the CMO libraries were made by performing 8 cycles of the indexing PCR using the supernatant. The transcriptome and CMO libraries were then sequenced by using either the Illumina NovaSeq or NextSeq machines


title: LIB5455518_SAM24414654_S1_L001

description:
SAM24414654_LIB5455518
NextSeq1