title: Treated 6 Male

status: Public on Apr 30 2025

city: Bloomington

first: Heather

last: O'Hagan

state: IN

address: 1001 E. 3rd St. Bloomington, IN 47405

institute: Indiana University

description:
Library name: Mouse_965
10X FLEX scRNAseq

data_processing:
Demultiplexing, alignment, and barcode processing , gene counting, and aggregation were made using Cellranger Multi Pipeline. (https://www.10xgenomics.com/support/software/cell-ranger/latest/analysis/running-pipelines/cr-3p-multi)
Assembly: Mouse mm10 Human GRC38
Supplementary files format and content: Tab seperated values files and matrix files

source_name: colon tumor

extract_protocol:
For sample preparation, 20-25 mg of fresh TP KO tumors (7 vehicle and 8 BRAFi+EGFRi) or 817 (3 vehicle and 3 BRAFi+EGFRi) were processed using the Tissue Fixation and Dissociation for Chromium Fixed RNA Profiling protocol (CG000553, 10X Genomics) and the Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit (1000414, 10X Genomics).
Gene expression libraries were prepared at the Indiana University School of Medicine (IUSM) Center for Medical Genomics using the Chromium Fixed RNA Kit, Mouse Transcriptome (1000497, 10x Genomics), in accordance with the user guide (Chromium Fixed RNA Profiling, CG000527- RevD). Briefly, 8,000 cells per sample were targeted for Gel-Beads-in-Emulsion (GEMs) formation after hybridization and pooling equally. The final library was generated and then sequenced using an Illumina NovaSeq X plus with approximately 2500 million reads.

value: colon tumor

value: TP KO organoids

value: Tumor Epithelial Cells

tag: treatment

value: Encorafenib + Gefitinib


title: HK2-TEAD1-rep1_S8

status: Public on May 28 2025

type: SRA

city: Baltimore

first: Danfeng

last: Cai

address: 615 N Wolfe St

country: USA

institute: Johns Hopkins Bloomberg School of Public Health

description: Library name: HK2-TEAD1-rep1_S8

library_source: genomic

data_processing:
ChIP-seq data were processed using the following pipeline. Raw sequencing reads in FASTQ format were quality-checked using FastQC. Reads were aligned to the human reference genome (hg38) using Bowtie2 (v2.3.5.3). The resulting BAM files were used to generate coverage tracks (bigWig format) using bamCoverage from the deepTools suite, without additional normalization.
Assembly: hg38
Supplementary files format and content: .bigwig file

molecule: genomic DNA

organism: Homo sapiens

source_name: kidney; Cortex; Proximal tubule

extract_protocol:
The cells, washed in ice-cold PBS, were then lysed in cell lysis buffer, with subsequent nutation at 4°C. Cells were treated by dounce homogenization on ice, followed by centrifuge at 500 x g for 10min. The supernatant was discarded, and the pellets was resuspended using nuclear lysis buffer. Nuclear lysates were then sheared by Covaris E220 to achieve average chromatin length of 300-400 bp.
DNA was purified with ChIP DNA Clean & ConcentratorTM (ZYMO research, D5205) and library preparation was performed using NEBNext® Multiplex Oligos for Illumima®

tag: tissue

value: kidney; Cortex; Proximal tubule

tag: cell line

tag: cell type

tag: antibody


title: Treated 3 Female

description:
Library name: Mouse_964
10X FLEX scRNAseq

library_source: transcriptomic single cell

molecule: total RNA

organism: Mus musculus


title: Treated 8 Male

description:
Library name: Mouse_977
10X FLEX scRNAseq


title: Treated 7 Male

description:
Library name: Mouse_967
10X FLEX scRNAseq


title: UOK122-TEAD1-rep1_S6

description: Library name: UOK122-TEAD1-rep1_S6

source_name: kidney

value: kidney

value: patient-derived cell line


title: Treated 4 Male

description:
Library name: Mouse_0
10X FLEX scRNAseq


title: Treated 2 Female

description:
Library name: Mouse_975
10X FLEX scRNAseq


title: Treated 5 Male

description:
Library name: Mouse_959
10X FLEX scRNAseq


title: Treated 1 Female

description:
Library name: Mouse_963
10X FLEX scRNAseq


title: Control 7 Male

description:
Library name: Mouse_983
10X FLEX scRNAseq

value: Vehicle


title: Control 1 Female

description:
Library name: Mouse_968
10X FLEX scRNAseq


title: Control 5 Male

description:
Library name: Mouse_978
10X FLEX scRNAseq


title: Control 4 Male

description:
Library name: Mouse_960
10X FLEX scRNAseq


title: Control 3 Female

description:
Library name: Mouse_979
10X FLEX scRNAseq


title: Control 6 Male

description:
Library name: Mouse_969
10X FLEX scRNAseq


title: Control 2 Female

description:
Library name: Mouse_966
10X FLEX scRNAseq


title: Camkk2+/+/Soft 1/IL4 1

city: Madison

first: Jianhong

last: Ou

state: Wisconsin

address: 330 N Orchard Street

institute: Morgridge Institute For Research

description: HWI-ST1276 (identifier)

library_source: transcriptomic

data_processing:
RNA-seq reads were trimmed by Trim Galore (v 0.6.7) and counted using salmon (v 1.4.0) for UCSC mm10 annotation.
Assembly: mm10
Supplementary files format and content: csv file of salmon's estimate of the relative abundance of this transcript in units of Transcripts Per Million (TPM)

molecule: polyA RNA

source_name: bone marrow

growth_protocol: Bone marrow (BM) cells were collected from the femur and tibiae of WT and Camkk2-/- male and female C57Bl/6 mice (8-10 weeks). The BM suspensions were passed through a 70 mm strainer and the red blood cells were lysed using RBC lysis buffer . The BM cells were collected and adjusted to 2´10e6/mL in DMEM conditional media (10% FBS and 30% L929 conditioned medium). BMs were plated in ultra-low attachment multi-well plates at 2´10e6/well. On day 4, half of the medium was refreshed and the cells were ready to use.

extract_protocol:
RNA was extracted using TRIzol reagent and purified by RNeasy mini kit. RNA was extracted and purified as described above. RNA from 2 biological replicates for each experimental point was used for RNA sequencing by Novogene Inc. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand of cDNA was synthesized using random hexamer primers. The second strand cDNA was synthesized using dUTP, instead of dTTP. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification.
mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers. Then the second strand cDNA was synthesized using dUTP, instead of dTTP. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.rara

treatment_protocol: The collagen-hyaluronic acid hydrogel solution was prepared and neutralized. BMDMs were resuspended in DMEM and mixed with the hydrogel solution at a final concentration of 4´106/mL. Dynamic stiffening was induced 24 hours after cell encapsulation by UV crosslinking using Irgacure 2959 as a photo initiator. After stiffening, the cells were cultured for 48 hours. Then, the hydrogel was digested by collagenase IV (400 U/mL) at 37°C for 1 hour. The detached BMDM were collected and processed for RNA extraction.

value: bone marrow

value: bone marro-derived macrophage

tag: genotype

value: WT

value: soft hydrogel/IL4


title: Camkk2+/+/Soft 1/IL4 2

description: HWI-ST1276 (identifier)


title: Camkk2+/+ /Soft 2

description: HWI-ST1276 (identifier)

value: soft hydrogel


title: Camkk2+/+ Stiff 2

description: HWI-ST1276 (identifier)

value: stiff hydrogel


title: Camkk2+/+ Stiff 1

description: HWI-ST1276 (identifier)


title: Camkk2+/+ /Soft 1

description: HWI-ST1276 (identifier)


title: P56_KO_YL13

status: Public on Apr 18 2025

city: Los Angeles

first: Genevieve

last: Konopka

state: CA

address: 650 Charles Young Drive South, 73-214 CHS

department: Neurobiology

institute: UCLA David Geffen School of Medicine

description: transcriptome

data_processing:
10X Genomics CellRanger v3.0.2
Seurat v3 and v4
Supplementary files format and content: raw counts matrix for filtered cells/nuclei in H5AD format

source_name: frontal cortex

extract_protocol:
Nuclei isolation protocol was modified from our previous work (Ayhan et al., 2021). Since homogenization of mouse cortex generated much fewer debris, we just did twice fltering through 40-μm Flowmi Cell Strainer (#H13680-0040, Bel-Art) without doing sucrose-gradient centrifugation
The single-nuclei RNA-seq libraries were prepared using Chromium Single Cell 3ʹ Reagent Kits v3 (CG000185 Rev B) from 10x Genomics

tag: strain

tag: age

value: KO

tag: gender

value: M

tag: rna preparation batch

tag: library preparation batch

tag: sequencing batch


title: Camkk2-/-/Soft 1

description: HWI-ST1276 (identifier)


title: P56_HU_YL05

description: transcriptome

value: HU


title: Camkk2-/-/Soft 1/IL4 1

description: HWI-ST1276 (identifier)


title: Camkk2-/-/Soft 2

description: HWI-ST1276 (identifier)


title: Camkk2-/- Stiff 1

description: HWI-ST1276 (identifier)


title: P56_KO_YL03

description: transcriptome


title: P56_HU_YL09

description: transcriptome


title: P56_HU_YL11

description: transcriptome

value: F


title: Camkk2-/- Stiff 2

description: HWI-ST1276 (identifier)


title: P56_KO_YL04

description: transcriptome


title: Camkk2-/-/Soft 1/IL4 2

description: HWI-ST1276 (identifier)


title: P56_KO_YL07

description: transcriptome


title: D1_C10_RNAseq

status: Public on May 31 2025

city: Cambridge

first: Laura

last: Biggins

address: Babraham Research Campus

department: Bioinformatics

country: United Kingdom

institute: The Babraham Institute

description: 2i_release_NMTSeq_RNAseq_filtered_normalized.txt

data_processing:
All sequencing was carried out on HiSeq instrument. RNA-seq libraries were pooled as 96-plex or 192-plex pools and sequenced using 75-bp paired-end reads.
scRNA-seq alignments were performed using Hisat 2 as described previously (Clark et al. Nature Communications, 2018).
Genome_build: GRCm38
Supplementary_files_format_and_content: Comma-delimited file with Ensembl Gene IDs, quantified values show number of reads per gene per sample

growth_protocol: E14 mESC were cultured in 2i+LIF media consisting out of N2B27 medium, composed out of DMEM/F12 (Life Technologies, 12634010) and Neurobasal (Life Technologies, 21103049) in a volume/volume (v/v) ratio 1:1, 0.1 mM 2-mercaptoethanol, 2 mM L-Glutamine, 1:200 v/v N2 (Life Technologies, 17502048) and 1:100 v/v B27 supplement (Life Technologies, 17504044), supplemented with 3 µM CHIR99021, 1 µM PD0325901 and 20 ng/ml of leukemia inhibitory factor (LIF)  (all Department of Biochemistry, University of Cambridge). mESCs were cultured on tissue culture plastic pre-coated with 0.1 % gelatine in H2O. Cultures were passaged when approaching confluence (every 2 days). All cells were cultured in a humidified incubator at 37 °C in 5 % CO2 and 20 % O2. mESCs were subject to routine mycoplasma testing using the MycoAlert testing kit (Lonza) and tested negative. For induction for 2i-relase experiment, the cell been rinsed with PBS before the culture media was changed to serum + LIF medium consisting of DMEM (Life Technologies, 10566-016), containing 15% Fetal Bovine Serum (FBS) (Gibco, 10270106), 1x non-essential amino acids (NEAA) (Life Technologies, 11140050), 0.1 mM 2-mercaptoethanol (Life Technologies, 31350-010), 2 mM L-Glutamine (Life Technologies, 25030-024), supplemented with 20 ng/ml of LIF (Department of Biochemistry, University of Cambridge).

extract_protocol:
0h, 24h, and 48h after induction of 2i-release  the cells been dissociated into single cells using accutase before flow sorting  (BD Influx) into 96-well plate containing 2.5μl of methylase reaction buffer (1 × M.CviPI Reaction buffer (NEB), 2 U M.CviPI (NEB), 160 μM S-adenosylmethionine (NEB), 1 U μl−1 RNasein (Promega), 0.1% IGEPAL CA-630 (Sigma)). Samples were incubated for 15 min at 37 °C to methylate accessible chromatin before the reaction was stopped with the addition of RLT plus buffer (Qiagen) and samples frozen down and stored at −80 °C before processing.
All downstream library preparation steps been performed as previously described by Clark et al. (Clark et al. Nature Communications, 2018).

value: embryonic stem cell


title: P07_KO_YL18

description: transcriptome


title: P07_KO_YL09

description: transcriptome


title: P07_KO_YL06

description: transcriptome


title: P07_HU_YL03

description: transcriptome


title: D1_G06

data_processing:
All sequencing was carried out on HiSeq instrument. BS-seq libraries were sequenced in 96-plex pools using 125-bp paired-end reads. RNA-seq libraries were pooled as 96-plex or 192-plex pools and sequenced using 75-bp paired-end reads.
Alignments of the single-cell bisulfite sequencing were performed using Bismark as well as subsequent CpG methylation and GpC accessibility calling.
Genome_build: GRCm38
Supplementary_files_format_and_content: The Bismark CpG coverage report is tab-delimited, uses 1-based genomic coordinates for every covered cytosine position in the experiment and is in the following format: <chromosome> <start position> <end position> <methylation percentage> <count methylated> <count non-methylated>

extract_protocol:
0h, 24h, and 48h after induction of 2i-release  the cells been dissociated into single cells using accutase before flow sorting  (BD Influx) into 96-well plate containing 2.5μl of methylase reaction buffer (1 × M.CviPI Reaction buffer (NEB), 2 U M.CviPI (NEB), 160 μM S-adenosylmethionine (NEB), 1 U μl−1 RNasein (Promega), 0.1% IGEPAL CA-630 (Sigma)). Samples were incubated for 15 min at 37 °C to methylate accessible chromatin before the reaction was stopped with the addition of RLT plus buffer (Qiagen) and samples frozen down and stored at −80 °C before processing.
E14 mESC were cultured in 2i+LIF media. For induction for 2i-relase experiment, the cell been rinsed with PBS before the culture media was changed to serum + LIF medium. 0h, 24h, and 48h after induction of 2i-release single cells were distracted and sc-NMT sequencing was performed.


title: P07_HU_YL17

description: transcriptome


title: D1_F11


title: P07_KO_YL01

description: transcriptome


title: D1_E10


title: P07_HU_YL12

description: transcriptome


title: P07_HU_YL05

description: transcriptome


title: P07_WT_YL16

description: transcriptome


title: P07_WT_YL11

description: transcriptome


title: P07_WT_YL04

description: transcriptome


title: P07_WT_YL02

description: transcriptome


title: D2_G11


title: AC_270_R10

status: Public on Apr 03 2025

city: Camperdown

first: G.

middle: Greg

last: Neely

state: NSW

address: Charles Perkins Centre, D17.

country: Australia

institute: University of Sydney

description: Isolated transgenes from Round 10 (E10) of Nb139-VP64 VLPs, to identify transgene substitutions.

data_processing:
Raw FASTQ files were aligned to transgene reference sequences (FASTA) with minimap2. Any mate-pair issues were fixed in the output .sam files with the samtools fixmate command before reads were sorted (samtools sort) and reformatted (samtools mpileup). VarScan 2.3.9 was used to call for variants with command mpileup2snp with arguments --min-coverage 10000 --min-reads2 15 --min-var-freq 0.00025 --p-value 0.01. Further annotation was performed with vcf-annotator using default commands, aligning to transgene reference sequences (GenBank format).
Assembly: ATGCAAGTGCAGCTTCAAGAATCCGGGGGCGGATTGGTTCAAGCCGGTGGTAGCCTCAGGCTTTCTTGTGCTGCATCCGAACGCACCTTCTCAACCTATGCTATGGGGTGGTTTCGGCAGGCCCCTGGTAGAGAGAGGGAGTTCCTTGCACAAATTAATTGGTCAGGGACAACTACATATTATGCCGAGTCTGTCAAGGATAGAACAACTATTTCTAGGGACAACGCCAAGAATACAGTTTATTTGGAAATGAATAACCTGAATGCTGACGATACAGGAATTTACTTTTGTGCAGCCCATCCACAGCGCGGCTGGGGTTCTACTTTGGGTTGGACCTACTGGGGGCAGGGAACTCAAGTAAGCTCCGTAAGTTCCGGTGGTGGGGGTTCAGGAGGGGGAAAGCCTATACCAAATCCACTGTTGGGCCTGGACTCAACTCGTACCGGACATCATCATCATCACCACTCAGGAAGTGGGAAGCGTCCTGCTGCTACAAAAAAAGCTGGACAGGCTAAGAAAAAAAAAGGCTCTGATGCACTGGATGATTTTGATCTCGATATGCTGGGAAGCGATGCCCTTGATGACTTTGATCTGGACATGCTGGGATCCGATGCTCTTGACGACTTCGACTTGGATATGTTGGGGTCCGATGCACTTGATGATTTTGACCTTGACATGCTGTAA
Supplementary files format and content: .vcf file (openable in Excel) containing analysis of mutation calls.
Library strategy: Amplicon sequencing

organism: Semliki Forest virus

extract_protocol:
Total RNA from VLP supernatant extracted. RT-PCR to amplify transgene sequences (amplicons used for library construction).
Samples were sequenced with Novogene using their ‘Microbial PCR Product Whole Genome Sequencing’ strategy. Amplicons underwent fragmentation prior to PCR-free library preparation and sequencing using NovaSeq PE250 technology.


title: AC_270_R15

description: Isolated transgenes from Round 15 (E15) of Nb139-VP64 VLPs, to identify transgene substitutions.


title: AC_270_R5

description: Isolated transgenes from Round 5 (E5) of Nb139-VP64 VLPs, to identify transgene substitutions.


title: AC_213_R10_2

description: Isolated transgenes from Round 10 (E10) of tTA VLPs, to identify transgene substitutions. [Campaign 1]

data_processing:
Raw FASTQ files were aligned to transgene reference sequences (FASTA) with minimap2. Any mate-pair issues were fixed in the output .sam files with the samtools fixmate command before reads were sorted (samtools sort) and reformatted (samtools mpileup). VarScan 2.3.9 was used to call for variants with command mpileup2snp with arguments --min-coverage 10000 --min-reads2 15 --min-var-freq 0.00025 --p-value 0.01. Further annotation was performed with vcf-annotator using default commands, aligning to transgene reference sequences (GenBank format).
Assembly: ATGGCTAGATTAGATAAAAGTAAAGTGATTAACagcgcattagagctgcttaatgaggtcggaatcgaaggtttaacaacccgtaaactcgcccagaagctaggtgtagagcagcctacattgtattggcatgtaaaaaataagcgggctttgctcgacgccttagccattgagatgttagataggcaccatactcacttttgccctttagaaggggaaagctggcaagattttttacgtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatggagcaaaagtacatttaggtacacggcctacagaaaaacagtatgaaactctcgaaaatcaattagcctttttatgccaacaaggtttttcactagagaatgcattatatgcactcagcgctgtggggcattttactttaggttgcgtattggaagatcaagagcatcaagtcgctaaagaagaaagggaaacacctactactgatagtatgccgccattattacgacaagctatcgaattatttgatcaccaaggtgcagagccagccttcttattcggccttgaattgatcatatgcggattagaaaaacaacttaaatgtgaaagtgggtccgcgtacagccgcgcgcgtacgaaaaacaattacgggtctaccatcgagggcctgctcgatctcccggacgacgacgcccccgaagaggcggggctggcggctccgcgcctgtcctttctccccgcgggacacacgcgcagactgtcgacggcccccccgaccgatgtcagcctgggggacgagctccacttagacggcgaggacgtggcgatggcgcatgccgacgcgctagacgatttcgatctggacatgttgggggacggggattccccgggtccgggatttaccccccacgactccgccccctacggcgctctggatatggccgacttcgagtttgagcagatgtttaccgatgcccttggaattgacgagtacggtgggtag
Supplementary files format and content: .vcf file (openable in Excel) containing analysis of mutation calls.
Library strategy: Amplicon sequencing


title: AC_270_R34

description: Isolated transgenes from Round 34 (E34) of Nb139-VP64 VLPs, to identify transgene substitutions.


title: AC_270_R26

description: Isolated transgenes from Round 26 (E26) of Nb139-VP64 VLPs, to identify transgene substitutions.


title: AC_213_R2_3

description: Isolated transgenes from Round 2 (E2) of tTA VLPs, to identify transgene substitutions. [Campaign 2]


title: AC_213_R8_2

description: Isolated transgenes from Round 8 (E8) of tTA VLPs, to identify transgene substitutions. [Campaign 1]


title: AC_270_R20

description: Isolated transgenes from Round 20 (E20) of Nb139-VP64 VLPs, to identify transgene substitutions.


title: AC_213_R6_3

description: Isolated transgenes from Round 6 (E6) of tTA VLPs, to identify transgene substitutions. [Campaign 2]


title: E2_20mol

description:
Library name: E2_20mol
Isolated transgenes from Round 2 of eGFP-LUC VLVs cultured in 20 uM molnupiravir for Round 1 and Round 2, to identify transgene substitutions.

data_processing:
Raw FASTQ files were aligned to transgene reference sequences (FASTA) with minimap2. Any mate-pair issues were fixed in the output .sam files with the samtools fixmate command before reads were sorted (samtools sort) and reformatted (samtools mpileup). VarScan 2.3.9 was used to call for variants with command mpileup2snp with arguments --min-coverage 10000 --min-reads2 15 --min-var-freq 0.00025 --p-value 0.01. Further annotation was performed with vcf-annotator using default commands, aligning to transgene reference sequences (GenBank format).
Assembly: ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTCAGGATCCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGGCCCATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTACGTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTGTCGACTGTTTAA
Supplementary files format and content: .vcf file (openable in Excel) containing analysis of mutation calls.

extract_protocol:
Total RNA from VLV supernatant extracted. RT-PCR to amplify transgene sequences (amplicons used for library construction).
Samples were sequenced with Novogene using their ‘Microbial PCR Product Whole Genome Sequencing’ strategy. Amplicons underwent fragmentation prior to PCR-free library preparation and sequencing using NovaSeq PE250 technology.


title: AC_270_RA

description: Isolated transgenes from Round A (amplification) of Nb139-VP64 VLPs, to identify transgene substitutions.


title: AC_213_R4_3

description: Isolated transgenes from Round 4 (E4) of tTA VLPs, to identify transgene substitutions. [Campaign 2]


title: AC_213_R10_3

description: Isolated transgenes from Round 10 (E10) of tTA VLPs, to identify transgene substitutions. [Campaign 2]


title: AC_213_R8_3

description: Isolated transgenes from Round 8 (E8) of tTA VLPs, to identify transgene substitutions. [Campaign 2]


title: AC_213_R6_2

description: Isolated transgenes from Round 6 (E6) of tTA VLPs, to identify transgene substitutions. [Campaign 1]


title: AC_213_R4_2

description: Isolated transgenes from Round 4 (E4) of tTA VLPs, to identify transgene substitutions. [Campaign 1]


title: AC_213_R2_2

description: Isolated transgenes from Round 2 (E2) of tTA VLPs, to identify transgene substitutions. [Campaign 1]


title: AC_150_R1

description: Isolated transgenes from Round 2 of eGFP-LUC VLPs, to identify transgene substitutions.

data_processing:
Raw FASTQ files were aligned to transgene reference sequences (FASTA) with minimap2. Any mate-pair issues were fixed in the output .sam files with the samtools fixmate command before reads were sorted (samtools sort) and reformatted (samtools mpileup). VarScan 2.3.9 was used to call for variants with command mpileup2snp with arguments --min-coverage 10000 --min-reads2 15 --min-var-freq 0.00025 --p-value 0.01. Further annotation was performed with vcf-annotator using default commands, aligning to transgene reference sequences (GenBank format).
Assembly: ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTCAGGATCCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGGCCCATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTACGTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTGTCGACTGTTTAA
Supplementary files format and content: .vcf file (openable in Excel) containing analysis of mutation calls.
Library strategy: Amplicon sequencing


title: AC_211_R30

description: Isolated transgenes from Round 30 (E30) of rtTA-3G VLPs, to identify transgene substitutions.

data_processing:
Raw FASTQ files were aligned to transgene reference sequences (FASTA) with minimap2. Any mate-pair issues were fixed in the output .sam files with the samtools fixmate command before reads were sorted (samtools sort) and reformatted (samtools mpileup). VarScan 2.3.9 was used to call for variants with command mpileup2snp with arguments --min-coverage 10000 --min-reads2 15 --min-var-freq 0.00025 --p-value 0.01. Further annotation was performed with vcf-annotator using default commands, aligning to transgene reference sequences (GenBank format).
Assembly: ATGTCTAGACTGGACAAGAGCAAAGtcataaactctgctctggaattactcaatggagtcggtatcgaaggcctgacgacaaggaaactcgctcaaaagctgggagttgagcagcctaccctgtactggcacgtgaagaacaagcgggccctgctcgatgccctgccaatcgagatgctggacaggcatcatacccactcctgccccctggaaggcgagtcatggcaagactttctgcggaacaacgccaagtcataccgctgtgctctcctctcacatcgcgacggggctaaagtgcatctcggcacccgcccaacagagaaacagtacgaaaccctggaaaatcagctcgcgttcctgtgtcagcaaggcttctccctggagaacgcactgtacgctctgtccgccgtgggccactttacactgggctgcgtattggaggaacaggagcatcaagtagcaaaagaggaaagagagacacctaccaccgattctatgcccccacttctgaaacaagcaattgagctgttcgaccggcagggagccgaacctgccttccttttcggcctggaactaatcatatgtggcctggagaaacagctaaagtgcgaaagcggcgggccgaccgacgcccttgacgattttgacttagacatgctcccagccgatgcccttgacgactttgaccttgatatgctgcctgctgacgctcttgacgattttgaCCTTGACATGCTCCCCGGGTAA
Supplementary files format and content: .vcf file (openable in Excel) containing analysis of mutation calls.
Library strategy: Amplicon sequencing


title: AC_150_RA

description: Isolated transgenes from Round 1 of eGFP-LUC VLPs, to identify transgene substitutions.


title: AC_211_R15

description: Isolated transgenes from Round 15 (E15) of rtTA-3G VLPs, to identify transgene substitutions.


title: AC_211_R25

description: Isolated transgenes from Round 25 (E25) of rtTA-3G VLPs, to identify transgene substitutions.


title: AC_211_RA

description: Isolated transgenes from Round A (amplification) of rtTA VLPs, to identify transgene substitutions.


title: D1R2_B

description:
Library name: D1R2_B
Isolated transgenes from Round 2 of tTA VLVs without selection, to identify transgene substitutions in ADAR/ADARB1 double KO cells

data_processing:
Raw FASTQ files were aligned to transgene reference sequences (FASTA) with minimap2. Any mate-pair issues were fixed in the output .sam files with the samtools fixmate command before reads were sorted (samtools sort) and reformatted (samtools mpileup). VarScan 2.3.9 was used to call for variants with command mpileup2snp with arguments --min-coverage 10000 --min-reads2 15 --min-var-freq 0.00025 --p-value 0.01. Further annotation was performed with vcf-annotator using default commands, aligning to transgene reference sequences (GenBank format).
Assembly: ATGGCTAGATTAGATAAAAGTAAAGTGATTAACagcgcattagagctgcttaatgaggtcggaatcgaaggtttaacaacccgtaaactcgcccagaagctaggtgtagagcagcctacattgtattggcatgtaaaaaataagcgggctttgctcgacgccttagccattgagatgttagataggcaccatactcacttttgccctttagaaggggaaagctggcaagattttttacgtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatggagcaaaagtacatttaggtacacggcctacagaaaaacagtatgaaactctcgaaaatcaattagcctttttatgccaacaaggtttttcactagagaatgcattatatgcactcagcgctgtggggcattttactttaggttgcgtattggaagatcaagagcatcaagtcgctaaagaagaaagggaaacacctactactgatagtatgccgccattattacgacaagctatcgaattatttgatcaccaaggtgcagagccagccttcttattcggccttgaattgatcatatgcggattagaaaaacaacttaaatgtgaaagtgggtccgcgtacagccgcgcgcgtacgaaaaacaattacgggtctaccatcgagggcctgctcgatctcccggacgacgacgcccccgaagaggcggggctggcggctccgcgcctgtcctttctccccgcgggacacacgcgcagactgtcgacggcccccccgaccgatgtcagcctgggggacgagctccacttagacggcgaggacgtggcgatggcgcatgccgacgcgctagacgatttcgatctggacatgttgggggacggggattccccgggtccgggatttaccccccacgactccgccccctacggcgctctggatatggccgacttcgagtttgagcagatgtttaccgatgcccttggaattgacgagtacggtgggtag
Supplementary files format and content: .vcf file (openable in Excel) containing analysis of mutation calls.

extract_protocol:
Total RNA from VLV supernatant extracted. RT-PCR to amplify transgene sequences (amplicons used for library construction).
Samples were sequenced with Australian Genomics Research Facility using their ‘Amplicon’ strategy. Amplicons underwent fragmentation prior to PCR-free library preparation and sequencing using NovaSeq PE250 technology.


title: AC_211_R20

description: Isolated transgenes from Round 20 (E20) of rtTA-3G VLPs, to identify transgene substitutions.


title: AC_150_RP

description: Isolated transgenes from Round P (packaging) of eGFP-LUC VLPs, to identify transgene substitutions.


title: AC_211_R10

description: Isolated transgenes from Round 10 (E10) of rtTA-3G VLPs, to identify transgene substitutions.


title: AC_213_RA

description: Isolated transgenes from Round A (amplification) of tTA VLPs, to identify transgene substitutions.


title: D1R2_A

description:
Library name: D1R2_A
Isolated transgenes from Round 2 of tTA VLVs without selection, to identify transgene substitutions in ADAR/ADARB1 double KO cells


title: D5R2_A

description:
Library name: D5R2_A
Isolated transgenes from tTA VLVs, to identify pre-existing mutations in ADAR/ADARB1 double KO cells


title: D5R2_B

description:
Library name: D5R2_B
Isolated transgenes from tTA VLVs, to identify pre-existing mutations in ADAR/ADARB1 double KO cells


title: B1R2_B

description:
Library name: B1R2_B
Isolated transgenes from Round 2 of tTA VLVs without selection, to identify transgene substitutions in ADARB1 KO cells


title: B1R2_A

description:
Library name: B1R2_A
Isolated transgenes from Round 2 of tTA VLVs without selection, to identify transgene substitutions in ADARB1 KO cells


title: B5R2_A

description:
Library name: B5R2_A
Isolated transgenes from tTA VLVs, to identify pre-existing mutations in ADARB1 KO cells


title: B5R2_B

description:
Library name: B5R2_B
Isolated transgenes from tTA VLVs, to identify pre-existing mutations in ADARB1 KO cells


title: A1R2

description:
Library name: A1R2
Isolated transgenes from Round 2 of tTA VLVs without selection, to identify transgene substitutions in ADAR KO cells


title: A5R2

description:
Library name: A5R2
Isolated transgenes from tTA VLVs, to identify pre-existing mutations in ADAR KO cells


title: E2_DMSO

description:
Library name: E2_DMSO
Isolated transgenes from Round 2 of eGFP-LUC VLVs cultured in DMSO for Round 1 and Round 2, to identify transgene substitutions.


title: F_SEQ026

description: Isolated transgenes from Round A (amplification) of co-packaged SRF-VP64/eGFP-LUC VLPs, to observe transgene proportion.

data_processing:
Up to 40 fmol of each DNA library was loaded on ONT Flongle flow cells (R9.4.1 chemistry, #FLO-FLG001) in a MinION Mk1B Sequencer (ONT, #MIN-101B) fitted with a Flongle Adapter (ONT, #ADP-FLG001). Sequencing was performed using MinKNOW (ONT, version 4.2.8) using default parameters and the following inputs: kit used, SQK-LSK110; 0.5 hours between MUX scans; basecalling, disabled. A minimum 2 x 104 raw reads were obtained per flow cell. Raw FAST5 files were basecalled using Guppy (version 4.5.2) with the minimum q-score filter set to 7.0. Quality-filtered basecalled reads (in FASTQ format) were processed using EPI2ME Desktop Agent (ONT, version 3.3.0.1031). Sequence reference files were uploaded into the program with the Fasta Reference Upload workflow (v2021.07.15). For each sample, all reads were aligned to reference files using the Fastq Custom Alignment workflow (v2021.03.25) using default parameters.
Assembly: ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTCAGGATCCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGGCCCATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTACGTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTGTCGACTGTTTAA; ATGCTCCCTACCCAGGCTGGCGCTGCTGCCGCTCTGGGCAGAGGCTCTGCTCTGGGAGGCTCTCTGAACCGGACCCCTACCGGCAGACCTGGAGGCGGCGGCGGCACCCGGGGCGCCAACGGAGGCAGAGTGCCTGGCAATGGCGCTGGTCTGGGCCCAGGCCGGCTGGAAAGAGAGGCCGCTGCCGCCGCCGCCACAACCCCTGCTCCAACCGCCGGCGCTCTGTATAGCGGATCCGAGGGCGATTCCGAGTCTGGCGAGGAAGAGGAACTGGGCGCTGAGCGGAGAGGCCTGAAGAGATCCCTGTCTGAGATGGAAATCGGCATGGTGGTGGGCGGACCTGAGGCCTCCGCTGCTGCTACAGGAGGCTACGGCCCTGTGTCTGGCGCCGTGTCCGGCGCCAAGCCTGGCAAGAAAACCCGCGGAAGAGTCAAGATCAAGATGGAATTCATCGACAACAAGCTGAGAAGATACACCACCTTCTCCAAGAGGAAAACAGGCATCATGAAGAAGGCCTACGAGCTGTCTACCCTGACAGGCACCCAGGTGCTGCTGCTGGTGGCTTCTGAGACTGGACACGTGTACACCTTTGCCACACGGAAACTGCAACCTATGATCACCTCTGAAACCGGAAAGGCCCTGATCCAGACCTGCCTGAATTCTCCAGATTCCCCTCCTAGATCTGATCCTACCACAGATCAGCGGATGTCTGCCACCGGATTTGAGGAAACCGACCTGACCTACCAAGTGTCCGAGTCCGACTCCTCTGGCGAGACAAAGGACACCCTCAAGCCATCAGGAAGTGGGAAGCGTCCTGCTGCTACAAAAAAAGCTGGACAGGCTAAGAAAAAAAAAGGCTCTGATGCACTGGATGATTTTGATCTCGATATGCTGGGAAGCGATGCCCTTGATGACTTTGATCTGGACATGCTGGGATCCGATGCTCTTGACGACTTCGACTTGGATATGTTGGGGTCCGATGCACTTGATGATTTTGACCTTGACATGCTGTAA; ATGAAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGGGGTGAATTGCAAGTTCACCATAGTTTTTCCACACAACCAAAAAGGAAACTGGAAAAATGTTCCTTCTAATTACCATTATTGCCCGTCAAGCTCAGATTTAAATTGGCATAATGACTTAATAGGCACAGCCTTACAAGTCAAAATGCCCAAGAGTCACAAGGCTATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAATGGGTCACTACTTGTGATTTCCGCTGGTATGGACCGAAGTATATAACACATTCCATCCGATCCTTCACTCCATCTGTAGAACAATGCAAGGAAAGCATTGAACAAACGAAACAAGGAACTTGGCTGAATCCAGGCTTCCCTCCTCAAAGTTGTGGATATGCAACTGTGACGGATGCCGAAGCAGTGATTGTCCAGGTGACTCCTCACCATGTGCTGGTTGATGAATACACAGGAGAATGGGTTGATTCACAGTTCATCAACGGAAAATGCAGCAATTACATATGCCCCACTGTCCATAACTCTACAACCTGGCATTCTGACTATAAGGTCAAAGGGCTATGTGATTCTAACCTCATTTCCATGGACATCACCTTCTTCTCAGAGGACGGAGAGCTATCATCCCTGGGAAAGGAGGGCACAGGGTTCAGAAGTAACTACTTTGCTTATGAAACTGGAGGCAAGGCCTGCAAAATGCAATACTGCAAGCATTGGGGAGTCAGACTCCCATCAGGTGTCTGGTTCGAGATGGCTGATAAGGATCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAGAAGGGTCAAGTATCTCTGCTCCATCTCAGACCTCAGTGGATGTAAGTCTAATTCAGGACGTTGAGAGGATCTTGGATTATTCCCTCTGCCAAGAAACCTGGAGCAAAATCAGAGCGGGTCTTCCAATCTCTCCAGTGGATCTCAGCTATCTTGCTCCTAAAAACCCAGGAACCGGTCCTGCTTTCACCATAATCAATGGTACCCTAAAATACTTTGAGACCAGATACATCAGAGTCGATATTGCTGCTCCAATCCTCTCAAGAATGGTCGGAATGATCAGTGGAACTACCACAGAAAGGGAACTGTGGGATGACTGGGCACCATATGAAGACGTGGAAATTGGACCCAATGGAGTTCTGAGGACCAGTTCAGGATATAAGTTTCCTTTATACATGATTGGACATGGTATGTTGGACTCCGATCTTCATCTTAGCTCAAAGGCTCAGGTGTTCGAACATCCTCACATTCAAGACGCTGCTTCGCAACTTCCTGATGATGAGAGTTTATTTTTTGGTGATACTGGGCTATCCAAAAATCCAATCGAGCTTGTAGAAGGTTGGTTCAGTAGTTGGAAAAGCTCTATTGCCTCTTTTTTCTTTATCATAGGGTTAATCATTGGACTATTCTTGGTTCTCCGAGTTGGTATCCATCTTTGCATTAAATTAAAGCACACCAAGAAAAGACAGATTTATACAGACATAGAGATGAACCGACTTGGAAAGTA
Supplementary files format and content: .xlsx file containing analysis of mapped read proportions.
Library strategy: Amplicon sequencing

extract_protocol:
Total RNA from VLP supernatant extracted. RT-PCR to amplify transgene sequences (amplicons used for library construction).
Sequencing samples were prepared following the recommendations found within the ONT protocol ‘Amplicons by Ligation’ (version ACDE_9110_v110_revV_10Nov2020). DNA concentrations were determined using the Qubit™ dsDNA HS Assay Kit (ThermoFisher, #Q32851) and 200 fmol of DNA was processed using the NEBNext Companion Module for ONT Ligation Sequencing (NEB, #E7180). Sequencing adapters were ligated onto sequences with the Ligation Sequencing Kit (ONT, #SQK-LSK110).


title: F_SEQ027

description: Isolated transgenes from Round E1 of co-packaged SRF-VP64/eGFP-LUC VLPs, to observe transgene proportion.


title: F_SEQ029

description: Isolated transgenes from Round E3 of co-packaged SRF-VP64/eGFP-LUC VLPs, to observe transgene proportion.


title: F_SEQ028

description: Isolated transgenes from Round E2 of co-packaged SRF-VP64/eGFP-LUC VLPs, to observe transgene proportion.


title: D2_D11


title: HUES64_Unlabeled

status: Public on Mar 05 2025

city: Irvine

first: Annie

last: Trinh

address: 419 S. Circle View Drive

department: Microbiology & Molecular Genetics

institute: University of California, Irvine

description: Library name: Unlabeled

data_processing:
For BrdU-labeled samples loaded on R9.4.1 flow cells (Oxford Nanopore Technologies, FLO-MIN106D), basecalling on raw current signals was performed using Guppy v6.5.7. DNA sequences were aligned to the hg19 human reference genome using Minimap2 v2.24 and chimeric reads were removed using samtools v1.15.1. To identify BrdU-containing reads, DNAscent v3.1.254 was used to detect the BrdU base analog within individual reads. CpG methylation was called using nanopolish v0.13.255. For BrdU-labeled samples loaded on R10.4.1 flow cells (Oxford Nanopore Technologies, FLO-MIN114) and unlabeled samples loaded on R10.4.1 flow cells (Oxford Nanopore Technologies, FLO-PRO114M), basecalling and alignment to the hg19 human reference genome were performed using Dorado v0.6.2. Chimeric reads were removed using samtools v1.15.1. To identify BrdU-containing reads, DNAscent v4.0.1 was used to detect the nucleoside analog within individual reads. CpG methylation was called using Dorado v0.6.2. Nanoplot v1.41.6 was used to assess and visualize read-level statistics such as number of reads, read length N50, mean and median read length, and mean and median read quality.
Assembly: GRCh37 (hg19)
Supplementary files format and content: For nanopore long-read samples, processed data are available in bedMethyl files (.bed) and read-level calls (.tsv). For Repli-ATAC samples, processed data are provided as .bigWig files.

growth_protocol: HUES64 hESCs were cultured on Geltrex (Thermo Fisher Scientific, A1413302) coated tissue culture plates with mTeSR (STEMCELL Technologies) media. Cells were incubated at 37°C with 5% CO2. Media was changed daily and cells were passaged upon reaching 80% confluency using ReleSR (STEMCELL Technologies).

extract_protocol:
For samples labeled with 50 µM BrdU or nocodazole, cells were lysed via incubation in SDS-PK buffer (0.5% wt/vol) sodium dodecyl sulfate (SDS), 50mM Tris-HCl, 0.01M EDTA, 1M NaCl and 0.2mg/ml proteinase K) at 56°C for 2h. Genomic DNA was isolated using phenol-chloroform extraction and isopropanol precipitation. For samples labeled with 500 µM BrdU, genomic DNA was isolated using the Puregene Cell Kit (8 x 10^8) (Qiagen, 158043) or Monarch® Genomic DNA Purification Kit (NEB, T3010S) according to the manufacturer’s instructions.
For samples labeled with 50 µM BrdU or nocodazole, libraries were prepared from purified and fragmented genomic DNA using a Ligation Sequencing Kit (Oxford Nanopore Technologies, SQK-LSK110) according to the manufacturer’s instructions. Briefly, DNA repair and end-prep were performed using NEBNext® FFPE DNA Repair Mix (NEB, M6630) and NEBNext® Ultra II End Repair/dA-tailing Module (NEB, E7546). Adapter ligation and clean-up were performed using the NEBNext® Quick Ligation Module (NEB, E6056). Long Fragment Buffer from the Ligation Sequencing Kit was used in the clean-up step to enrich for DNA fragments of >3 kb. Libraries were loaded onto R9.4.1 flow cells (Oxford Nanopore Technologies, FLO-MIN106D) and sequenced using a MinION Mk1b device (Oxford Nanopore Technologies) for 16 hours. Minimum read length was set to 200 bp. For samples labeled with 500 µM BrdU and samples not labeled with BrdU, libraries were prepared using a Ligation Sequencing Kit (Oxford Nanopore Technologies, SQK-LSK114) according to the manufacturer’s instructions, similarly to previously described. Libraries for samples labeled with 500 µM BrdU were loaded onto R10.4.1 flow cells (Oxford Nanopore Technologies, FLO-MIN114) and sequenced using a MinION Mk1b device for 32 hours. Libraries generated from unlabeled samples were loaded onto R10.4.1 flow cells (Oxford Nanopore Technologies, FLO-PRO114M) and sequenced on a PromethION P2 solo device (Oxford Nanopore Technologies) for 72 hours. Minimum read length was set to 1000 bp. All sequencing data was collected using MinKNOW v23.07.5.

treatment_protocol: For the nocodazole experiment, hESCs were treated with 2 µg/ml nocodazole for 16h prior to DNA extraction. For the BrdU pulse-chase experiments, BrdU pulses are conducted by adding BrdU to the culture medium at the desired final concentration and incubating for the specified durations (10 minutes, 20 minutes, 30 minutes, or 1 hour). Cells are then either collected immediately (0h chase) or washed three times with DPBS and incubated in fresh mTeSR medium for 16 hours (16h chase). For mock treatments, BrdU is replaced with an equivalent volume of Ultrapure H2O. Cells subjected to mock treatments undergo the same incubation times as their BrdU-treated counterparts, with samples collected at 0h or following a 16h incubation.

value: human embryonic stem cells

value: Unlabeled (no BrdU)


title: AC_190_191

description: RNA from HEK293T cells expressing eGFP, to identify RT-PCR-generated substitutions.

extract_protocol:
Total cellular RNA from HEK293T cells expressing eGFP extracted. RT-PCR to amplify eGFP sequences (amplicons used for library construction).
Samples were sequenced with Novogene using their ‘Microbial PCR Product Whole Genome Sequencing’ strategy. Amplicons underwent fragmentation prior to PCR-free library preparation and sequencing using NovaSeq PE250 technology.


title: F_SEQ025

description: Isolated transgenes from Round P (packaging) of co-packaged SRF-VP64/eGFP-LUC VLPs, to observe transgene proportion.


title: H1299_rna_sgM5_R4

status: Public on Mar 01 2025

city: nanjing

first: Zhou

last: Jun

state: jiangsu

address: longmiandadao

department: College of life science and technology

country: China

institute: China Pharmaceutical University

data_processing:
For paired-end sequencing data, only read 2 from the raw FASTQ files was used for subsequent analyses. Adapter sequences and low-quality bases were trimmed from ribosome-protected fragment (RPF) samples using Cutadapt (v2.8) with the following parameters: -a AGATCGGAAGAGCGT -u 10 -q 20 -e 0.1 -O 6 -m 25 -M 35 --discard-untrimmed. For the corresponding input samples, the minimum fragment length parameter was set to -m 20, without applying an upper fragment length limit (-M). To remove rRNA and tRNA contamination, reads were aligned to human rRNA and tRNA reference sequences using Bowtie2 (v2.2.5), and unmapped reads were retained as clean reads. These clean reads were then aligned to the human reference genome (GRCh38) using HISAT2 (v2.2.1) with the -k 1 parameter. Gene-level read counts were quantified using featureCounts (v2.0.1) with the -t exon parameter, based on the GRCh38.106 GTF annotation file from the Ensembl database. The protein-coding genes were retained for downstream analysis. To correct for batch effects across different datasets, the ComBat_seq function from the R package sva (v3.58.0) was applied to the count matrix. The batch-corrected count data were then used for differential translation efficiency (TE) analysis. TE differences were calculated using Riborex (v2.4.0) with the edgeR (v4.8.0) engine. Genes with an absolute fold change ≥ 1.5 and a p-value < 0.05 were considered significantly regulated at the translational level. Downstream quality control of Ribo-seq data was performed using the riboTransVis (v0.99.0) R package, focusing on the 5’end of reads.
Assembly: GRCh38
Supplementary files format and content: Comma-separated values (CSV) file with gene counts

source_name: lung

growth_protocol: A549 and H1299 lung cancer cells were cultured in DMEM (BI, Cat#06-1055-57-1ACS) and RPMI 1640(BI, Cat#01-100-1ACS) supplemented with 10% FBS (Viva Cell, C04001-500) and 1% Penicillin-Streptomycin (Life,15140122) at 37℃ with 5% CO2.

extract_protocol:
Input RNA was extracted with TRIzol LS followed by chemical fragmentation. Ribo-seq samples were produced by RNase I digestion followed by RNA extraction using TRIzol LS.
40-60nt region for input and 30nt for ribo-seq were selected according to 15% polyacrylamide TBE-urea gel separation. Desired RNA species were excised and soaked overnight followed by ethanol precipitation. The purified RNA was dephosporylated and added with 3’adapter. The 5’ adapter was added after reverse transcription. Eventually, PCR amplification was proceeded using Phusion High-Fidelity DNA Polymerase.

treatment_protocol: A549 with H1299 lung cancer cells are treated with fresh complete medium for 2 h

value: lung

value: Non-Small Cell Lung Cancer

value: METTL5 knockout

value: treated with CHX for 5min,normal culture


title: H1299_ribo_sgM5_R3

molecule: other


title: H1299_rna_sgM5_R3


title: H1299_rna_sgCtrl_R2


title: H1299_ribo_sgCtrl_R2

data_processing:
Assembly: GRCh38
Supplementary files format and content: Comma-separated values (CSV) file with gene counts


title: H1299_rna_sgM5_R1


title: A549_rna_sgM5_R1


title: H1299_ribo_sgM5_R4


title: H1299_ribo_sgM5_R1


title: A549_rna_sgM5_R4


title: AC_pCDH150

description: Isolated transgenic sequence from pSFV-DE_eGFP-LUC plasmid DNA, sequenced to control for identification of transgene substitutions in VLPs.

library_source: other

organism: synthetic construct

source_name: synthetic construct

extract_protocol:
Maxiprepped plasmid DNA. RT-PCR to amplify transgene sequences (amplicons used for library construction).
Samples were sequenced with Novogene using their ‘Microbial PCR Product Whole Genome Sequencing’ strategy. Amplicons underwent fragmentation prior to PCR-free library preparation and sequencing using NovaSeq PE250 technology.

value: synthetic construct


title: AC_211_R5

description: Isolated transgenes from Round 5 (E5) of rtTA-3G VLPs, to identify transgene substitutions.


title: A549_rna_sgM5_R3


title: A549_rna_sgCtrl_R2


title: A549_ribo_sgM5_R4


title: SD, replicate 3, snRNA-seq

status: Public on Feb 10 2025

description:
10X Genomics
sleepseq_matrix.mtx.gz
sleepseq_barcodes.tsv.gz
sleepseq_features.tsv.gz
sleepseq_meta.tsv.gz

data_processing:
CellRanger mkfastq, CellRanger count
FastQC
CellBender
DoubletFinder
Seurat
Assembly: mm10 (GRCm38p6)
Supplementary files format and content: sleepseq_matrix.mtx.gz' has raw counts for all samples.
Supplementary files format and content: sleepseq_barcodes.tsv.gz' has list of barcodes for all samples matching with raw counts.
Supplementary files format and content: sleepseq_features.tsv.gz' has list of genes matching to raw counts.
Supplementary files format and content: sleepseq_meta.tsv.gz' has all meta information including assigned celltypes.

molecule: nuclear RNA

source_name: Brain, Cortex

extract_protocol:
Nuclei for snRNA-seq were isolated from mice, 8-10 weeks old.
Droplet-based snRNA-seq libraries were prepared using Chromium Single Cell 3ʹ v3.1 (1000121, 10x Genomics) according to the manufacturer’s protocol42. Libraries were sequenced using an Illumina NovaSeq 6000.

treatment_protocol: All mice were accommodated for 1-2 weeks to a cage and treadmill, consis7ng of a bofomless plexiglass enclosure suspended over the treadmill with ad lib access to food and water. For sleep depriva7on (SD) the slow-moving treadmill (0.1KM/hour) was engaged from ZT 0-6 hours and for recovery sleep (RS) from ZT 0-4. There was no treadmill engagement for RS from ZT 4-6 hours and for control sleep (CS), no treadmill engagement for the en7re ZT 0-6 hours period. All animals were sacrificed at ZT=6 hours and 7ssue rapidly prepared for either electrophysiological (exVivo brain slices) or transcriptomic analysis

value: Brain, Cortex

value: wild-type

value: SD


title: SD, replicate 4, snRNA-seq


title: A549_ribo_sgCtrl_R2

value: WT


title: A549_ribo_sgM5_R3


title: SD, replicate 1, snRNA-seq


title: CS, replicate 3, snRNA-seq

value: CS


title: SD, replicate 2, snRNA-seq


title: CS, replicate 4, snRNA-seq


title: CS, replicate 2, snRNA-seq


title: A549_ribo_sgM5_R1


title: AO24

status: Public on Feb 07 2025

description: 10x Genomics

data_processing:
cellranger mkfastq
cellranger count
cellbender
scrublet
doubletFinder
seurat
Assembly: mm10
Supplementary files format and content: barcodes.tsv' a tab-delimited text files include list of cell-barcodes and 'features.tsv' a tab-delimited text file include list of genes and 'matrix.mtx' file in a sparse matrix format containing raw counts per gene per cell-barcode.

source_name: NEOCORTEX

extract_protocol:
Single nuclei were obtained from cortex samples in 1 ml ice cold Nuclei EZ lysis buffer (Sigma, #EZ PREP NUC-101), homogenized using 2 ml glass dounce tissue grinders on ice; 15 times with pestle A and then 15 times with pestle B. The solution was then transferred to a new 2 mL microcentrifuge tube, 1mL nuclei EZ lysis buffer was added, followed by 5-minute incubation on ice, and then centrifuged at 500xg for 5 minutes at 4°C. Supernatants were removed, pellets were resuspended in 1mL nuclei EZ lysis buffer, followed by an additional 5-minute incubation on ice, and centrifuged at 500xg for 5 minutes at 4°C. The supernatant was then discarded, the pellet resuspended in NSB (1X PBS w/ 0.2U/ul RNase inhibitors /1% Ultra-pure BSA) and filtered through a 40um Flowmi Cell strainer (Bel-Art, H13680-0040). Nuclei were then counted on a hemocytometer (iChip) in a 10ul of suspension with 1:1 0.4% Trypan Blue. Nuclei concentration was adjusted for targeted sequencing of 10,000 cells/sample using the 10X Genomics Single Cell 3′ Reagent Kits v3 protocol to prepare libraries. 4 mice/genotype and timepoint were used, using both males and females. Libraries were sequenced using the McDermott Sequencing Core at UT Southwestern.
Library was performed according to the manufacter’s instructions (single cell 3’ v3 10x Genomics). Isolated nuclei were mixed with the 10X  master mix and loaded with partitioning oil and gel beads onto the provided chip. This results in generation of gel bead-in-emulsion (GEMs).  GEMs were then retrotranscripted to cDNA; these contained  Ilumina R1 primer sequence, Unique Molecular Identifier (UMIs) and the 10x Barcode. Silane DynaBeads were used to purify cDNA which were then amplified by PCR. SPRIselect beads were used to select proper sized fragments. Samples were cleaned with 80% ethanol after every amplification step as well. Finally, libraries were constructed and an Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added during these final steps.

value: NEOCORTEX

tag: batch prep

tag: sequencing batch_cumulative

value: FLX


title: CS, replicate 1, snRNA-seq


title: AO23

description: 10x Genomics


title: AO22

description: 10x Genomics

value: CKO


title: AO18

description: 10x Genomics


title: AO19

description: 10x Genomics


title: AO20

description: 10x Genomics


title: AO17

description: 10x Genomics


title: AO14

description: 10x Genomics


title: AO13

description: 10x Genomics


title: AO21

description: 10x Genomics


title: AO10

description: 10x Genomics


title: AO16

description: 10x Genomics


title: AO09

description: 10x Genomics


title: AO11

description: 10x Genomics


title: AO15

description: 10x Genomics


title: AO8

description: 10x Genomics


title: AO12

description: 10x Genomics


title: AO7

description: 10x Genomics


title: AO4

description: 10x Genomics


title: AO1

description: 10x Genomics


title: P0_REP3

description: 10x Genomics

data_processing:
cellranger-atac mkfastq
cellranger-atac count
signac,macs2 (peak generation)
signac
Assembly: mm10
Supplementary files format and content: Respective files differentiated by age in name:'barcodes.tsv.gz' a tab-delimited text files include list of cell-barcodes and 'features.tsv.gz' a tab-delimited text file include list of peaks and 'matrix.mtx.gz' file in a sparse matrix format containing raw counts per peak per cell-barcode.

source_name: Neocortex

extract_protocol:
Nuclei were isolated per modification of Habib et al. 2017(Habib et al. 2017) and Ayhan et al. 2021(Ayhan et al. 2021) protocol. Cortex samples were placed in a 2mL dounce homogenizer on ice with 1 mL ice cold Nuclei EZ lysis buffer (Sigma, #EZ PREP NUC-101), and homogenized; 15-20 times with pestle A and then 15-20 times with pestle B. The solution was then transferred to a clean 2 mL microcentrifuge tube, 1mL nuclei EZ lysis buffer was added, followed by 4-minute incubation on ice, and centrifugation at 500xg (rcf) for 5 minutes at 4°C. The supernatant was then discarded, pellet resuspended in 300uL-1mL Nuclei Wash Buffer (Tris-HCl pH7.4 10mM, NaCl 10mM, MgCl2 3mM, BSA 10%, Tween-20 1%), then centrifuged 500xg for 5 min at 4C. The supernatant was discarded, and pellet resuspended in chilled diluted Nuclei Buffer (10X genomics) by adding 100uL diluted Nuclei Buffer on ice to allow for natural dissociation for 3-4min. Then the final volume was brought to 300uL-1mL. This was then filtered through a 40um pre-wetted Flowmi Cell strainer into a 1.5mL microcentrifuge tube. Nuclei were then counted on a hemocytometer (iChip) in a 10ul of suspension with 1:1 0.4% Trypan Blue. Nuclei concentration was adjusted for targeted sequencing of 4,000-7,000 nuclei/sample using the 10X Genomics Chromium Single Cell ATAC v1.1-v2 protocol to prepare libraries. 2-3 mice/genotype and timepoint were used, using both males and females. Libraries were sequenced using the McDermott Sequencing Core at UT Southwestern.
Library was performed according to the manufacter’s instructions (single cell ATAC v1.1 and v2 protocol, 10x Genomics). Isolated nuclei were mixed with the transposition mix which was followed by transposition. 10X master mix added and samples were loaded onto chip alongside  partitioning oil and gel beads. This results in generation of gel bead-in-emulsion (GEMs).  GEMs were then cleaned with Silane DynaBeads  followed by size-selection using SPRIselect beads. Samples were cleaned with 80% ethanol. Finally, libraries were constructed with  paired-end constructs with P5 and P7 sequences and a sample index added during the final steps.

value: Neocortex

tag: prep batch

value: e1 cKO


title: AO3

description: 10x Genomics


title: AO6

description: 10x Genomics


title: AO5

description: 10x Genomics


title: P0_REP2

description: 10x Genomics


title: P0_REP6

description: 10x Genomics

value: flox control


title: P0_REP5

description: 10x Genomics


title: P0_REP1

description: 10x Genomics


title: E16_REP4

description: 10x Genomics


title: E16_REP1

description: 10x Genomics


title: E16_REP3

description: 10x Genomics


title: P0_REP4

description: 10x Genomics


title: E16_REP2

description: 10x Genomics


title: AO2

description: 10x Genomics


title: E13_REP6

description: 10x Genomics


title: E13_REP2

description: 10x Genomics


title: Brain, Mecp2 KO/+, exp3

status: Public on Jan 31 2025

type: RNA

city: St. Louis

first: Mati

middle: Tesfaye

last: Nemera

state: Missouri

address: 660 S Euclid Ave

department: Neuroscience

institute: Washington University in St. Louis School of Medicine

description: Vizgen MERSCOPE Instrument

hyb_protocol: Hybridization of the tissue to gene probes for MERFISH was performed according to instructions provided by Vizgen. Briefly, after aspiration of the 70% ethanol, the 10 μm-thick tissue section mounted onto the MERSCOPE slide was washed with Sample Prep Wash Buffer (Vizgen, PN 20300001) and Formamide Wash Buffer (Vizgen, PN 20300002) while placed in the petri dish, and incubated at 37°C for 30 min in a benchtop incubator (INCU-Line, VWR). The Formamide Wash Buffer was then aspirated, and 50 μL of a custom-designed MERSCOPE gene panel mix was delivered onto the tissue section. A 2 × 2 cm piece of Parafilm M (Fisher Scientific) was carefully placed onto the tissue so that the gene panel mix was evenly spread across the tissue and no bubbles were introduced between the tissue and the film. The petri dish was closed with a lid and sealed with Parafilm M, sterilized with 70% ethanol, and placed in a humidified benchtop incubator (INCU-Line, VWR) set at 37°C for 36–48 h.

data_processing:
Cells were segmented using the Watershed algorithm, using DAPI as the Seed Stain and PolyT as the Watershed Stain.
Single-cell expression matrices were produced from quantifying the number of RNA transcripts in segmented cells.
Cell-by-gene matrix csv files provide the RNA counts of each gene in each cell. Metadata csv files provide information about each cell such as cell volume and location. detected_transcripts.csv files include 3D locations of detected transcripts.
mecp2Het_exp_cell_annotations.csv file includes total SCTransform-corrected and raw transcript counts, cell volumes, transcriptotype, and cell taxonomy information for female Mecp2 heterozygous knockout mouse MERFISH experiments.
WT_exp_cell_annotations.csv file includes total SCTransform-corrected and raw transcript counts, cell volumes, transcriptotype, and cell taxonomy information for female wild-type mouse MERFISH experiments.

extract_protocol: Brains were placed in O.C.T-containing molds and flash frozen in liquid nitrogen. 10 μm-thick coronal tissue sections were prepared from the O.C.T. compound-embedded brain using a cryostat. Tissue sections were mounted onto specialized MERSCOPE slides (Vizgen, PN 20400001), and maintained in 70% ethanol in a sealed petri dish until gene probe hybridization according to Vizgen's protocol.

value: Brain

value: Mecp2 KO/+

value: Female


title: Brain, Mecp2 KO/+, exp2

description: Vizgen MERSCOPE Instrument


title: E13_REP3

description: 10x Genomics


title: Brain, Mecp2 KO/+, exp4

description: Vizgen MERSCOPE Instrument


title: NSCLC_KRAS_12_lpwgs

status: Public on Jan 17 2025

city: New York

first: Somnath

last: Tagore

state: NY

address: William Black Building, 17th Floor, Lab 1708E

institute: Columbia University

description: low pass whole genome sequencing

data_processing:
library strategy: low pass whole genome sequencing
fastq to sam conversion using bowtie2 v2.4.5
sam to bam conversion using samtools v1.16.1
bam to wig conversion using readCounter (hmmcopy_utils)
wig to seg file conversion using ichorCNA v0.2.0
Assembly: hg38
Supplementary files format and content: segementation file for CNA
Supplementary files format and content: ichorcna tumor cna prediction segmentation file
Supplementary files format and content: cna prediction segmentation file

source_name: NSCLC primary tumor

extract_protocol:
Nuclei from snRNA-seq sample preparations were collected by centrifugation (500g, 5 min), snap frozen after removing excess ST buffer, and stored at -20C until further processing. If insufficient nuclei (<100,000) were present, additional tissue curls were cut and processed in the same manner as for single-nuclei extraction. Genomic DNA was extracted from nuclei using DNeasy Blood and Tissue kit (Qiagen, catalog no.69504) according to manufacturer's instructions. DNA was quantified using a Nanodrop.
20-50 ng of genomic DNA was utilized for input for library prepation with Lotus DNA Library Prep Kit (Integrated DNA Technologies- IDT, Corlville, IA) using xGEN Stubby Adapter and UDI Primer pairs according to manufacturer's instructions with the folllowing specifications. During the enzymatic preparation program, sampless were held at 32C for 9 minutees. Adapters were not diluted, and the optional PCR (5 cycles) and cleanup were performed. After library cleanup with AMPure beads (Beckman Coulter, Brea, CA), libraries were quantified with Tapestation D5000 HS tapes (Agilent) and stored at -20C until sequencing

tag: metastatic site

value: PT

tag: patient id

tag: stk11 mutation

tag: kras mutation status

value: KRAS Q61H

tag: sequencing technique

value: lp-WGS

tag: tcr-seq

value: yes


title: E13_REP4

description: 10x Genomics

tag: tissue


title: NSCLC_KRAS_13_lpwgs

description: low pass whole genome sequencing

value: KRAS G12V


title: Brain, WT

description: Vizgen MERSCOPE Instrument

tag: genotype


title: Brain, Mecp2 KO/+, exp1

description: Vizgen MERSCOPE Instrument

tag: strain


title: E13_REP5

description: 10x Genomics

tag: age


title: E13_REP1

description: 10x Genomics


title: NSCLC_KRAS_11_lpwgs

description: low pass whole genome sequencing

value: KRAS G12D


title: NSCLC_KRAS_10_lpwgs

description: low pass whole genome sequencing

value: WT

value: KRAS G12C


title: NSCLC_KRAS_17_lpwgs

description: low pass whole genome sequencing

source_name: NSCLC brain metastasis

value: BM


title: NSCLC_KRAS_7_lpwgs

description: low pass whole genome sequencing


title: NSCLC_STK_1_lpwgs

description: low pass whole genome sequencing

value: STK11 deletion exons 2-5

value: KRAS G13C


title: NSCLC_N586_lpwgs

description: low pass whole genome sequencing


title: NSCLC_STK_20_lpwgs

description: low pass whole genome sequencing

value: KRAS WT


title: NSCLC_STK_21_lpwgs

description: low pass whole genome sequencing

value: STK11 c.169delG p.E57fs*


title: NSCLC_KRAS_6_lpwgs

description: low pass whole genome sequencing


title: AC_270_R30

description: Isolated transgenes from Round 30 (E30) of Nb139-VP64 VLPs, to identify transgene substitutions.

tag: cell line


title: NSCLC_KRAS_8_lpwgs

description: low pass whole genome sequencing


title: NSCLC_STK_3_lpwgs

description: low pass whole genome sequencing

value: STK11 splice site 863-1G>T


title: NSCLC_STK_15_lpwgs

description: low pass whole genome sequencing

value: STK11 p.P221Lfs


title: NSCLC_STK_22dot2_lpwgs

description: low pass whole genome sequencing


title: NSCLC_STK_18_lpwgs

description: low pass whole genome sequencing

value: STK11 Q305X


title: NSCLC_PA141_lpwgs

description: low pass whole genome sequencing


title: NSCLC_STK_5dot2_lpwgs

description: low pass whole genome sequencing

value: STK11 c.836delG, p.G279fs


title: NSCLC_STK_14_lpwgs

description: low pass whole genome sequencing


title: P56_HU_YL02

description: transcriptome

data_processing:
10X Genomics CellRanger v3.0.2
Seurat v3 and v4
Supplementary files format and content: raw counts matrix for filtered cells/nuclei in H5AD format

value: HU

tag: gender

value: F

tag: rna preparation batch


title: NSCLC_STK_5dot1_lpwgs

description: low pass whole genome sequencing

value: lp-WGS


title: D1_A03_RNAseq

description: 2i_release_NMTSeq_RNAseq_filtered_normalized.txt

data_processing:
All sequencing was carried out on HiSeq instrument. RNA-seq libraries were pooled as 96-plex or 192-plex pools and sequenced using 75-bp paired-end reads.
scRNA-seq alignments were performed using Hisat 2 as described previously (Clark et al. Nature Communications, 2018).
Genome_build: GRCm38
Supplementary_files_format_and_content: Comma-delimited file with Ensembl Gene IDs, quantified values show number of reads per gene per sample

tag: cell type

value: embryonic stem cell


title: D2_F10

data_processing:
All sequencing was carried out on HiSeq instrument. BS-seq libraries were sequenced in 96-plex pools using 125-bp paired-end reads. RNA-seq libraries were pooled as 96-plex or 192-plex pools and sequenced using 75-bp paired-end reads.
Alignments of the single-cell bisulfite sequencing were performed using Bismark as well as subsequent CpG methylation and GpC accessibility calling.
Genome_build: GRCm38
Supplementary_files_format_and_content: The Bismark CpG coverage report is tab-delimited, uses 1-based genomic coordinates for every covered cytosine position in the experiment and is in the following format: <chromosome> <start position> <end position> <methylation percentage> <count methylated> <count non-methylated>


title: NSCLC_PA125_lpwgs

description: low pass whole genome sequencing

value: yes


title: NSCLC_PA060_lpwgs

description: low pass whole genome sequencing


title: NSCLC_PA042_lpwgs

description: low pass whole genome sequencing


title: NSCLC_KRAS_12_slideseq

description: Single-nuclei RNA sequencing, SlideSeqV2

data_processing:
library strategy: single-nuclei RNAseq; SlideSeqV2
We used the Slide-seq tools pipeline (Stickels et al., 2021) to process our Slide-seq data, and loaded the data into Seurat.
Assembly: GRCh38
Supplementary files format and content: Counts data (*.counts.csv.gz) and Spatial coordinates data (*.coords.csv.gz)

extract_protocol:
RNA from 10 uM frozen tumor sections was extracted and spatially indexed as described in Stickels et al. (Nature Biotechnology, 2021). Briefly, sections were cut and then mounted on a "puck". RNA was hybridized for 15 min in 6x SCC and then reveres transcribed using Maxima RT.  Tissue was then digested and cleared using clearing buffer and proteinase K and unbound capture oligos were removed using EXO-I treatment. 2nd strand synthesis was then performed using sn-SMRT oligos and Klenow Enzyme and the libraries were amplified using PCR with Truseq and SMART PCR primers.
Libraries after PCR amplification were tagmented and indexed using Nextera XT kit and Nextera N70x oligos. After SPRI cleanup libraries were quantified using D5000 tapestation and prepared for sequencing.

value: Primary Tumor

tag: stk11 mutation status

value: KRAS Q61H

value: snRNA-seq, SlideSeqV2

value: no


title: NSCLC_PA072_lpwgs

description: low pass whole genome sequencing

value: BM


title: NSCLC_PA104_lpwgs

description: low pass whole genome sequencing


title: NSCLC_PA001_lpwgs

description: low pass whole genome sequencing


title: NSCLC_PA067_lpwgs

description: low pass whole genome sequencing


title: NSCLC_PA070_lpwgs

description: low pass whole genome sequencing


title: NSCLC_PA068_lpwgs

description: low pass whole genome sequencing


title: NSCLC_KRAS_11_slideseq

value: WT

value: KRAS G12D


title: NSCLC_PA054_lpwgs

description: low pass whole genome sequencing


title: NSCLC_PA043_lpwgs

description: low pass whole genome sequencing


title: NSCLC_PA080_lpwgs

description: low pass whole genome sequencing


title: NSCLC_PA004_lpwgs

description: low pass whole genome sequencing


title: NSCLC_PA048_lpwgs

description: low pass whole genome sequencing


title: NSCLC_PA025_lpwgs

description: low pass whole genome sequencing


title: NSCLC_PA005_lpwgs

description: low pass whole genome sequencing


title: NSCLC_PA056_lpwgs

description: low pass whole genome sequencing


title: NSCLC_KRAS_10_slideseq

value: KRAS G12C


title: NSCLC_STK22dot2_slideseq

value: KRAS WT


title: NSCLC_N586_slideseq


title: NSCLC_PA019_slideseq

value: Brain Metastasis


title: NSCLC_STK_18_slideseq

value: STK11 Q305X


title: NSCLC_STK_15_slideseq

value: STK11 p.P221Lfs


title: NSCLC_KRAS_12_tcr

description: Single-nuclei RNA sequencing

data_processing:
library strategy: single-cell RNAseq (TCR-seq)
Fastq files were analyzed using vdj in Cell Ranger (v6.1.1, 10x Genomics). Transcripts were aligned to the human GRCh38 reference genome.
Assembly: GRCh38
Supplementary files format and content: Cell Ranger vdj output files (csv)

extract_protocol:
Single-nuclei suspension from frozen NSCLC metastases were generated using methods published in Slyper et al. (Nature Medicine, 2020) with modifications. The tissue was first cut in 20 µm sections using a cryostat and stored on dry ice until the next step. Then the tissue was put on wet ice and washed once with 5 ml PBS to remove OCT. After this step nuclei were extracted by vigorous pipetting in salt-tris buffer with Tween-20 (TST)  followed by a high-volume wash in salt-tris buffer (ST). Nuclei were counted and 15,000 nuclei were separated for droplet-based RNA capture and reverse transcription using 10x Genomics  5' v1.1 or 5' V2.0  reagents and a Chromium controller.
Chromium Single Cell V(D)J Enrichment Kit for human T cells Reagent Kit, v 1.1 or Single Cell Human TCR Amplification Kit were utilized for snTCR library generation (10X genomics) depending on whether 5' V1 or 5' V2 kits were utilized for cDNA library generation, respectively. Final sequencing libraries were prepared using Single Index Kit T Set A or Library construction kit (catalog no. 1000190) and Dual Index Kit TT set A (catalog no. 1000215).

value: PT

value: snRNA-seq


title: NSCLC_PA025_slideseq


title: NSCLC_KRAS_10_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_KRAS_8_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_KRAS_11_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_STK_14_slideseq


title: NSCLC_KRAS_6_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_KRAS_17_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_PA056_slideseq


title: NSCLC_KRAS_7_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_STK_14_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_STK_21_tcr

description: Single-nuclei RNA sequencing

value: STK11 c.169delG p.E57fs*


title: NSCLC_STK_20_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_STK_2_tcr

description: Single-nuclei RNA sequencing

value: KRAS G12A


title: NSCLC_STK_5dot2_tcr

description: Single-nuclei RNA sequencing

value: STK11 c.836delG, p.G279fs

value: yes


title: NSCLC_STK_22dot2_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_N586_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_N254_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_STK_1_tcr

description: Single-nuclei RNA sequencing

value: STK11 deletion exons 2-5

value: KRAS G13C


title: NSCLC_STK_15_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_PA125_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_STK_3_tcr

description: Single-nuclei RNA sequencing

value: STK11 splice site 863-1G>T


title: NSCLC_STK_5dot1_tcr

description: Single-nuclei RNA sequencing

value: BM


title: NSCLC_PA141_tcr

description: Single-nuclei RNA sequencing


title: D0_B01

value: embryonic stem cell


title: NSCLC_PA104_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_PA072_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_PA080_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_PA068_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_PA076_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_PA067_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_PA056_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_PA060_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_PA042_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_PA034_tcr

description: Single-nuclei RNA sequencing


title: CaMKiiα Cre +/-; Mef2c fl/fl Treated 3

data_processing:
FastQC for raw read quality
Trimmomatic for trimming reads and adapters
STAR for read alignment to MM10 genome
DESeq2 analysis for DEGs
GO analysis using ToppFunn and SynGO
Assembly: MM10
Supplementary files format and content: Xlsx, CPM values

source_name: Brain

extract_protocol:
RNA extraction
150 bp paired end reads

value: Brain

value: CaMKiialpha Cre +/-; Mef2c fl/fl

tag: treatment

value: 100 uM Picrotoxin and 100 uM 4 Aminopyridine


title: NSCLC_PA043_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_PA025_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_PA054_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_PA019_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_PA005_tcr

description: Single-nuclei RNA sequencing


title: CaMKiiα Cre +/-; Mef2c fl/fl Treated 2


title: NSCLC_PA048_tcr

description: Single-nuclei RNA sequencing


title: CaMKiiα Cre +/-; Mef2c fl/fl Vehicle 3

value: 1% DMSO + 1% Water


title: NSCLC_PA004_tcr

description: Single-nuclei RNA sequencing


title: NSCLC_PA001_tcr

description: Single-nuclei RNA sequencing


title: CaMKiiα Cre +/-; Mef2c fl/fl Vehicle 1


title: CaMKiiα Cre +/-; Mef2c fl/fl Vehicle 2


title: NSCLC_PT11

description:
Single-nuclei RNA sequencing
KRAS_13

data_processing:
library strategy: single nuclei RNAseq
Raw snRNA-seq data were analyzed using Cell Ranger (v5.0.1). Transcripts were aligned to the human GRCh38 reference genome.
Ambient RNA was removed using CellBender (v0.2.0). Gene expression matrices were processed individually in R (v4.1.2) using Seurat (v4.0.5).
Filters were applied to keep cells with 500–10,000 genes, 1,000–60,000 UMIs, and <10% of mitochondrial reads in each individual sample.
Scrublet v0.2.1 (Wolock et al., 2019) was applied to identify and remove doublets with an expected doublet rate ranging from 2.8–6.4% based on the loading rate.
Assembly: GRCh38
Supplementary files format and content: mtx file (MatrixMarket format) of combined gene expression count matrix
Supplementary files format and content: csv file of gene names corresponding to the combined gene expression count matrix
Supplementary files format and content: csv file of integrated gene expression matrix

extract_protocol:
Single-nuclei suspension from frozen NSCLC metastases were generated using methods published in Slyper et al. (Nature Medicine, 2020) with modifications. The tissue was first cut in 20 µm sections using a cryostat and stored on dry ice until the next step. Then the tissue was put on wet ice and washed once with 5 ml PBS to remove OCT. After this step nuclei were extracted by vigorous pipetting in salt-tris buffer with Tween-20 (TST)  followed by a high-volume wash in salt-tris buffer (ST). Nuclei were counted and 15,000 nuclei were separated for droplet-based RNA capture and reverse transcription using 10x Genomics  5' v1.1 or 5' v2.0 reagents and a Chromium controller.
Single-nuclei RNAseq (snRNA-seq) libraries were generated using Chromium 5' (V1.1 and V2) reagents according to the manufacturer protocol with one additional cycle during library amplification to account for lower material input from nuclei compared to whole cells.

value: WT

value: KRAS G12V


title: Mef2c fl/fl Treated 3 (FLX_CAMK17_2_M_TREATED_S20)

value: Mef2c fl/fl


title: CaMKiiα Cre +/-; Mef2c fl/fl Treated 1


title: Mef2c fl/fl Treated 2


title: NSCLC_PT09

description:
Single-nuclei RNA sequencing
KRAS_11

value: PT

value: KRAS G12D

value: snRNA-seq


title: Mef2c fl/fl Treated 1


title: NSCLC_PT08

description:
Single-nuclei RNA sequencing
KRAS_10

value: KRAS G12C


title: NSCLC_PT10

description:
Single-nuclei RNA sequencing
KRAS_12

value: KRAS Q61H

value: yes


title: NSCLC_BM32

description:
Single-nuclei RNA sequencing
KRAS_17


title: NSCLC_PT05

description:
Single-nuclei RNA sequencing
STK_20

value: KRAS WT


title: NSCLC_BM30

description:
Single-nuclei RNA sequencing
KRAS_7


title: NSCLC_PT04

description:
Single-nuclei RNA sequencing
STK_15

value: STK11 p.P221Lfs


title: NSCLC_BM29

description:
Single-nuclei RNA sequencing
KRAS_6

value: BM


title: Mef2c fl/fl Vehicle 1


title: NSCLC_BM31

description:
Single-nuclei RNA sequencing
KRAS_8


title: NSCLC_PT06

description:
Single-nuclei RNA sequencing
STK_21

value: STK11 c.169delG p.E57fs*


title: Mef2c fl/fl Vehicle 3

value: Brain


title: NSCLC_PT07

description:
Single-nuclei RNA sequencing
STK_22dot2


title: NSCLC_PT03

description:
Single-nuclei RNA sequencing
STK_2

value: KRAS G12A


title: NSCLC_BM26

description:
Single-nuclei RNA sequencing
STK_14


title: NSCLC_BM27

description:
Single-nuclei RNA sequencing
STK_18

value: STK11 Q305X


title: Mef2c fl/fl Vehicle 2

value: 1% DMSO + 1% Water


title: NSCLC_BM25

description:
Single-nuclei RNA sequencing
STK_5dot2

value: STK11 c.836delG, p.G279fs


title: NSCLC_BM23

description:
Single-nuclei RNA sequencing
STK_3

value: STK11 splice site 863-1G>T

value: KRAS G13C


title: NSCLC_BM24

description:
Single-nuclei RNA sequencing
STK_5dot1


title: NSCLC_BM19

description:
Single-nuclei RNA sequencing
PA104


title: NSCLC_PT02

description:
Single-nuclei RNA sequencing
N586

value: PT


title: NSCLC_BM21

description:
Single-nuclei RNA sequencing
PA141


title: NSCLC_BM20

description:
Single-nuclei RNA sequencing
PA125


title: NSCLC_BM18

description:
Single-nuclei RNA sequencing
PA080


title: NSCLC_BM15

description:
Single-nuclei RNA sequencing
PA070


title: NSCLC_BM16

description:
Single-nuclei RNA sequencing
PA072


title: NSCLC_BM12

description:
Single-nuclei RNA sequencing
PA060


title: NSCLC_BM22

description:
Single-nuclei RNA sequencing
STK_1

value: STK11 deletion exons 2-5


title: NSCLC_BM11

description:
Single-nuclei RNA sequencing
PA056

value: snRNA-seq


title: NSCLC_BM17

description:
Single-nuclei RNA sequencing
PA076


title: NSCLC_BM14

description:
Single-nuclei RNA sequencing
PA068


title: NSCLC_BM13

description:
Single-nuclei RNA sequencing
PA067


title: NSCLC_PT01

description:
Single-nuclei RNA sequencing
N254


title: NSCLC_BM08

description:
Single-nuclei RNA sequencing
PA043


title: NSCLC_BM07

description:
Single-nuclei RNA sequencing
PA042


title: NSCLC_BM09

description:
Single-nuclei RNA sequencing
PA048


title: NSCLC_BM10

description:
Single-nuclei RNA sequencing
PA054


title: NSCLC_BM06

description:
Single-nuclei RNA sequencing
PA034


title: NSCLC_KRAS_13_tcr

description: Single-nuclei RNA sequencing

value: WT

value: KRAS G12V


title: NSCLC_BM03

description:
Single-nuclei RNA sequencing
PA005

value: yes


title: NSCLC_BM02

description:
Single-nuclei RNA sequencing
PA004


title: NSCLC_BM04

description:
Single-nuclei RNA sequencing
PA019


title: NSCLC_BM05

description:
Single-nuclei RNA sequencing
PA025


title: NSCLC_BM01

description:
Single-nuclei RNA sequencing
PA001


title: Retina,2.5mo,donor 2,snATAC

status: Public on Jan 15 2025

city: La Jolla

first: Ying

last: Yuan

address: Palmilla Dr.

department: MATS

institute: UCSD

description: 2.5 mo old wild type mice retinal tissue_Left eye dissected

library_source: genomic single cell

data_processing:
CellRanger
Assembly: mm10
Supplementary files format and content: CellRanger output files: atac_fragments.tsv.gz, atac_fragments.tsv.gz.tbi
Supplementary files format and content: bigWig files: Cell type-specific pseudo bulk ATAC-seq signal track

source_name: retina

extract_protocol:
The flash frozen retinal tissue along with RPE and choroid was harvested from 28days, 2.5month, 5month and 21month old mice. Each age point contained two biological replicates. Nuclei preparation was adapted from Lacar et al. 2016 (PMID: 27090946). Flash frozen mouse retina was resuspended in 1 mL of  douncing buffer consisting of 0.25M sucrose (S1888, Sigma), 25mM KCl (AM9610G, Invitrogen), 5mM MgCl2 (194698, Mp Biomedicals Inc.), 10 mM Tris-HCl pH 7.5 (15567027, Thermo Fisher Scientific), 1mM DTT (D9779, Sigma), 1X protease inhibitor (05056489001, Roche), 0.1% Triton X-100 (T8787-100ML, Sigma), and 0.5 U/ul RNasin RNase inhibitor (PAN21110, Promega) in molecular biology grade water (46000-CM, Corning). Frozen tissue was then transferred to a dounce homogenizer on ice and dounced 25 times with a loose plunger and 25 times with a tight plunger. Suspension was then passed through a Celltrix 30 μM filter (04-004-2326, Sysmex) and washed with 300 mL of douncing buffer before being centrifuged for 10 minutes at 1000 rcf in a swinging bucket centrifuge at 4°C with run setting 3/3. Pellet was then washed with an additional 1 mL of douncing buffer without Triton X-100, and split for single-nucleus ATAC-seq and single-nucleus RNA-seq in a ratio of 700 mL /300 mL.
Nuclei from dissociated tissue were permeabilized in 1 mL of  permeabilization buffer (5% BSA, 0.2% IGEPAL-CA630, 1 mM DTT, 1x cOmplete EDTA-free protease inhibitor in PBS), pipette mixed, and incubated on ice for 10 minutes. Nuclei were then centrifuged at 500 rcf for 5 minutes and the supernatant was removed. Nuclei were resuspended in 500 µL high salt tagmentation buffer (36.3 mM Tris-acetate (pH = 7.8), 72.6 mM potassium-acetate, 11 mM Mg-acetate, 17.6% DMF) and counted using a hemocytometer. Concentration was adjusted to 2000 nuclei/9 µL, and 2000 nuclei were dispensed into each well of one 96-well plate. For tagmentation, 1 µL barcoded Tn5 transposomes (58) was added using a BenchSmart 96 (Mettler Toledo), mixed five times and incubated for 60 min at 37°C with shaking (500 rpm). To inhibit the Tn5 reaction, 10 µL of 40 mM EDTA were added to each well with a BenchSmart 96 (Mettler Toledo) and the plate was incubated at 37°C for 15 min with shaking (500 rpm). Next, 10 µL 3x sort buffer (2% BSA, 2 mM EDTA in PBS) was added using a BenchSmart 96 (Mettler Toledo). All wells were combined into a FACS tube and stained with 3 µM Draq7 (Cell Signaling). Using a SH800 (Sony), 20 2n nuclei were sorted per well into eight 96-well plates (total of 768 wells) containing 10.5 µL EB (25 pmol) primer i7, 25 pmol primer i5, 200 ng BSA (Sigma). Preparation of sort plates and all downstream pipetting steps were performed on a Biomek i7 Automated Workstation (Beckman Coulter). After addition of 1 µL 0.2% SDS, samples were incubated at 55°C for 7 min with shaking (500 rpm). 1 µL 12.5% Triton-X was added to each well to quench the SDS. Next, 12.5 µL NEBNext High-Fidelity 2 × PCR Master Mix (NEB) were added and samples were PCR-amplified (72°C 5 min, 98°C 30 s, (98°C 10 s, 63°C 30 s, 72°C 60 s)×12 cycles, held at 12°C). After PCR, all wells were combined. Libraries were purified according to the MinElute PCR Purification Kit manual (Qiagen) using a vacuum manifold (QIAvac 24 plus, Qiagen) and size selection was performed with SPRI Beads (Beckmann Coulter, 0.55x and 1.5x). Libraries were purified one more time with SPRI Beads (Beckmann Coulter, 1.5x).

value: retina


title: NSCLC_PA034_lpwgs

description: low pass whole genome sequencing

value: lp-WGS


title: Retina,5mo,donor 1,snATAC

description: 5mo old wild type mice Left Eye


title: Retina,5mo,donor 2,snATAC

description: 5mo old wild type mice  Right Eye


title: Retina,2.5mo,donor 1,snATAC


title: resistant_rep2

first: Richard

middle: A

last: Young

state: MA

address: 9 Cambridge Center

institute: Whitehead Institute for Biomedical Research

description: rRNA depleted total RNA

data_processing:
Reads were mapped to the human genome GRCh38 using STAR aligner (v2.7.1a), allowing up to 100 multiple alignments and up to 200 loci anchors (--outFilterMultimapNmax 100 --winAnchorMultimapNmax 200).
Differential expression analysis of genes and transposable elements comparing triplicates of samples treated with normal or pathogenic concentrations of insulin was performed using TEtranscripts (v2.2.3).
Assembly: hg38
Supplementary files format and content: comma-separated values file contains differential expression data for annotated transcripts
Supplementary files format and content: cntTable.txt: raw counts
Supplementary files format and content: Rlog_annotated.csv: rlog-transformed counts

source_name: Liver

extract_protocol:
Cells were washed with EMEM and RNA was then purified using TRIzolTMreagent following manufacturer’s instructions.
RNA-seq libraries were prepared using a KAPA RNA HyperPrep kit with RiboErase. Spike-ins were included but were not used for analysis.

treatment_protocol: HepG2 cells were treated with 0.1nM (normal) or 3nM (pathogenic) insulin concentrations in EMEM 0.125% BSA for three days.

value: Liver

value: Hepatocellular carcinoma

value: 3 nM insulin


title: resistant_rep1

description: rRNA depleted total RNA


title: sensitive_rep3

description: rRNA depleted total RNA

value: 0.1 nM insulin


title: sensitive_rep1

description: rRNA depleted total RNA


title: sensitive_rep2

description: rRNA depleted total RNA


title: NSCLC_N254_slideseq

value: Primary Tumor

value: snRNA-seq, SlideSeqV2

value: no


title: NSCLC_PA054_slideseq

value: Brain Metastasis


title: F_SEQ030

description: Isolated transgenes from Round E4 of co-packaged SRF-VP64/eGFP-LUC VLPs, to observe transgene proportion.

data_processing:
Up to 40 fmol of each DNA library was loaded on ONT Flongle flow cells (R9.4.1 chemistry, #FLO-FLG001) in a MinION Mk1B Sequencer (ONT, #MIN-101B) fitted with a Flongle Adapter (ONT, #ADP-FLG001). Sequencing was performed using MinKNOW (ONT, version 4.2.8) using default parameters and the following inputs: kit used, SQK-LSK110; 0.5 hours between MUX scans; basecalling, disabled. A minimum 2 x 104 raw reads were obtained per flow cell. Raw FAST5 files were basecalled using Guppy (version 4.5.2) with the minimum q-score filter set to 7.0. Quality-filtered basecalled reads (in FASTQ format) were processed using EPI2ME Desktop Agent (ONT, version 3.3.0.1031). Sequence reference files were uploaded into the program with the Fasta Reference Upload workflow (v2021.07.15). For each sample, all reads were aligned to reference files using the Fastq Custom Alignment workflow (v2021.03.25) using default parameters.
Assembly: ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTCAGGATCCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGGCCCATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTACGTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTGTCGACTGTTTAA; ATGCTCCCTACCCAGGCTGGCGCTGCTGCCGCTCTGGGCAGAGGCTCTGCTCTGGGAGGCTCTCTGAACCGGACCCCTACCGGCAGACCTGGAGGCGGCGGCGGCACCCGGGGCGCCAACGGAGGCAGAGTGCCTGGCAATGGCGCTGGTCTGGGCCCAGGCCGGCTGGAAAGAGAGGCCGCTGCCGCCGCCGCCACAACCCCTGCTCCAACCGCCGGCGCTCTGTATAGCGGATCCGAGGGCGATTCCGAGTCTGGCGAGGAAGAGGAACTGGGCGCTGAGCGGAGAGGCCTGAAGAGATCCCTGTCTGAGATGGAAATCGGCATGGTGGTGGGCGGACCTGAGGCCTCCGCTGCTGCTACAGGAGGCTACGGCCCTGTGTCTGGCGCCGTGTCCGGCGCCAAGCCTGGCAAGAAAACCCGCGGAAGAGTCAAGATCAAGATGGAATTCATCGACAACAAGCTGAGAAGATACACCACCTTCTCCAAGAGGAAAACAGGCATCATGAAGAAGGCCTACGAGCTGTCTACCCTGACAGGCACCCAGGTGCTGCTGCTGGTGGCTTCTGAGACTGGACACGTGTACACCTTTGCCACACGGAAACTGCAACCTATGATCACCTCTGAAACCGGAAAGGCCCTGATCCAGACCTGCCTGAATTCTCCAGATTCCCCTCCTAGATCTGATCCTACCACAGATCAGCGGATGTCTGCCACCGGATTTGAGGAAACCGACCTGACCTACCAAGTGTCCGAGTCCGACTCCTCTGGCGAGACAAAGGACACCCTCAAGCCATCAGGAAGTGGGAAGCGTCCTGCTGCTACAAAAAAAGCTGGACAGGCTAAGAAAAAAAAAGGCTCTGATGCACTGGATGATTTTGATCTCGATATGCTGGGAAGCGATGCCCTTGATGACTTTGATCTGGACATGCTGGGATCCGATGCTCTTGACGACTTCGACTTGGATATGTTGGGGTCCGATGCACTTGATGATTTTGACCTTGACATGCTGTAA; ATGAAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGGGGTGAATTGCAAGTTCACCATAGTTTTTCCACACAACCAAAAAGGAAACTGGAAAAATGTTCCTTCTAATTACCATTATTGCCCGTCAAGCTCAGATTTAAATTGGCATAATGACTTAATAGGCACAGCCTTACAAGTCAAAATGCCCAAGAGTCACAAGGCTATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAATGGGTCACTACTTGTGATTTCCGCTGGTATGGACCGAAGTATATAACACATTCCATCCGATCCTTCACTCCATCTGTAGAACAATGCAAGGAAAGCATTGAACAAACGAAACAAGGAACTTGGCTGAATCCAGGCTTCCCTCCTCAAAGTTGTGGATATGCAACTGTGACGGATGCCGAAGCAGTGATTGTCCAGGTGACTCCTCACCATGTGCTGGTTGATGAATACACAGGAGAATGGGTTGATTCACAGTTCATCAACGGAAAATGCAGCAATTACATATGCCCCACTGTCCATAACTCTACAACCTGGCATTCTGACTATAAGGTCAAAGGGCTATGTGATTCTAACCTCATTTCCATGGACATCACCTTCTTCTCAGAGGACGGAGAGCTATCATCCCTGGGAAAGGAGGGCACAGGGTTCAGAAGTAACTACTTTGCTTATGAAACTGGAGGCAAGGCCTGCAAAATGCAATACTGCAAGCATTGGGGAGTCAGACTCCCATCAGGTGTCTGGTTCGAGATGGCTGATAAGGATCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAGAAGGGTCAAGTATCTCTGCTCCATCTCAGACCTCAGTGGATGTAAGTCTAATTCAGGACGTTGAGAGGATCTTGGATTATTCCCTCTGCCAAGAAACCTGGAGCAAAATCAGAGCGGGTCTTCCAATCTCTCCAGTGGATCTCAGCTATCTTGCTCCTAAAAACCCAGGAACCGGTCCTGCTTTCACCATAATCAATGGTACCCTAAAATACTTTGAGACCAGATACATCAGAGTCGATATTGCTGCTCCAATCCTCTCAAGAATGGTCGGAATGATCAGTGGAACTACCACAGAAAGGGAACTGTGGGATGACTGGGCACCATATGAAGACGTGGAAATTGGACCCAATGGAGTTCTGAGGACCAGTTCAGGATATAAGTTTCCTTTATACATGATTGGACATGGTATGTTGGACTCCGATCTTCATCTTAGCTCAAAGGCTCAGGTGTTCGAACATCCTCACATTCAAGACGCTGCTTCGCAACTTCCTGATGATGAGAGTTTATTTTTTGGTGATACTGGGCTATCCAAAAATCCAATCGAGCTTGTAGAAGGTTGGTTCAGTAGTTGGAAAAGCTCTATTGCCTCTTTTTTCTTTATCATAGGGTTAATCATTGGACTATTCTTGGTTCTCCGAGTTGGTATCCATCTTTGCATTAAATTAAAGCACACCAAGAAAAGACAGATTTATACAGACATAGAGATGAACCGACTTGGAAAGTA
Supplementary files format and content: .xlsx file containing analysis of mapped read proportions.
Library strategy: Amplicon sequencing