# This is a metadata template for submission of high-throughput sequencing samples to the GEO database.

# Fields with a red triangle in the upper-right corner have pop-up instructions that can be displayed if you hover over t

STUDY
# This section describes the overall study.
# Information provided in this section will be displayed in a GEO Series (GSE record) on public web pages.
title
summary (abstract)
summary (abstract)
experimental design
contributor
contributor
contributor
contributor
supplementary file optional

SAMPLES
# Information provided in this section will be displayed in GEO Samples (GSM records) on public web pages.
# A GEO Sample record will be created from each row in this section.
# Biological replicates of the same sample: If provided, should be listed on different rows and titled accordingly (biol r
# Technical replicates, eg, the same libraries were run in different lanes of a flow cell or sequenced multiple times: If p
library name
HEK293T_H2AZ_CST
HEK293T_H2AZ_ABCAM
HEK293T_H3_3_EMDMillipore
HEK293T_H4K16ac_CST
HEK293T_H3K79me2_CST
HEK293T_Input

PROTOCOLS
# Information provided in this section will appear in each GEO Sample (GSM record). These fields can be moved to the
# Please ensure that the annotations are relevant to the SAMPLES above (not to the samples from a previous submiss
growth protocol optional
treatment protocol optional
extract protocol
library construction protocol
library strategy

data processing step

data processing step
data processing step
data processing step
data processing step
data processing step
genome build/assembly
processed data files format and content

PAIRED-END EXPERIMENTS
# If "paired-end" experiments are included, list the files for each paired-end run in a row. Each row will become one se
# Single-cell data: If applicable, list index files (I1, I2, etc.) in "file name 3", "file name 4" columns.
# Please make sure all raw files listed here are also listed in the "raw file" columns in the above SAMPLES section.
file name 1
sequencing samples to the GEO database.
nstructions that can be displayed if you hover over the field.

GSE149484

Series (GSE record) on public web pages.

Decoding the Protein Composition of Whole Nucleosomes with Nuc-MS
Current proteomic approaches disassemble and digest nucleosome particles, blurring readouts of the ‘histone code’. To preser
Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone variants H3.3, H2AZ as well as the histone modification
Luis,F,Schachner
Marta,Iwanaszko
Marc,A,Morgan
Ali,Shilatifard
Neil,Kelleher

mples (GSM records) on public web pages.

listed on different rows and titled accordingly (biol rep 1, biol rep 2, and so on).
lanes of a flow cell or sequenced multiple times: If provided, list all raw files in the same row, adding more "raw file" columns as
title organism cell line
HEK293T, H2AZ, CST Homo sapiens HEK293T
HEK293T, H2AZ, ABCAM Homo sapiens HEK293T
HEK293T, H3, 3, EMDMillipore Homo sapiens HEK293T
HEK293T, H4K16ac, CST Homo sapiens HEK293T
HEK293T, H3K79me2, CST Homo sapiens HEK293T
HEK293T, Input Homo sapiens HEK293T

mple (GSM record). These fields can be moved to the above SAMPLES section in order to provide different information for differe
S above (not to the samples from a previous submission).
DMEM, 15% fetal bovine serum, 2mM glutamine, 1x penicillin/streptomycin. Cells were cultured at 37C at 5% CO2.

Cells were fixed with 1% formaldehyde for 10 minutes. After quenching and cell lysis, chromatin was sonicated using a E220 fo
ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes fro
ChIP-Seq

Basecalls were performed using bcl2fastq v2.17 for Novaseq output.

ChIP-seq reads were trimmed from 3' end until the final base had a quality score > 30, using Trimmomatic v0.33, discarding rea
ChIP-seq reads were aligned to the UCSC hg38 genome using Bowtie version 1.1.2. Only uniquely mapping reads with at mos
To create ChIP-seq coverage plots, the locations of the mapped ChIP-seq reads were extended to 150 bp to represent sequen
Peaks were called uing MACS v2.1.0 with the significance cut-off q-value <=0.01
bigWig files were generated using the genomicRanges R package. Score represents the normalized coverage of DNA fragmen
 hg38
bigWig, narrowPeak (except for Input sample)

aired-end run in a row. Each row will become one sequencing run on a GEO Sample (GSM record).
name 3", "file name 4" columns.
aw file" columns in the above SAMPLES section.
file name 2
eadouts of the ‘histone code’. To preserve nucleosome-level information, we developed Nuc-MS, which displays the landscape of histone va
H2AZ as well as the histone modifications H3K79me2 and H4K16ac in HEK293T cells.

, adding more "raw file" columns as needed to accommodate all raw files.
cell type ChIP antibody molecule
Human embryonic kidney H2AZ (CST, #2718S) Genomic DNA
Human embryonic kidney H2AZ (Abcam, #ab4174) Genomic DNA
Human embryonic kidney H3.3 (EMD/Millipore, #09-838) Genomic DNA
Human embryonic kidney H4K16ac (CST, #13534S) Genomic DNA
Human embryonic kidney H3K79me2 (CST, #5427S) Genomic DNA
Human embryonic kidney none Genomic DNA

ovide different information for different samples.

cultured at 37C at 5% CO2.

hromatin was sonicated using a E220 focused-ultrasonicator (Covaris).

ented with NEXTflex DNA Barcodes from Bioo Scientific. 10 ng of DNA was used as starting material for input and ip samples. Libraries we

using Trimmomatic v0.33, discarding reads left with < 20 bp

nly uniquely mapping reads with at most two mismatches were retained.
xtended to 150 bp to represent sequenced fragments, renormalized (to reads per million, rpm) and reformatted in the bigWig file format.

e normalized coverage of DNA fragments at a given genomic coordinate. narrowPeak files were generated using MACS v2 with default sett
S, which displays the landscape of histone variants and their post-translational modifications (PTMs) in a single mass spectrum. Combined

single or paired-end instrument model description

single Illumina NovaSeq 6000
single Illumina NovaSeq 6000
single Illumina NovaSeq 6000
single Illumina NovaSeq 6000
single Illumina NovaSeq 6000
single Illumina NovaSeq 6000

aterial for input and ip samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected

and reformatted in the bigWig file format.

e generated using MACS v2 with default settings.

odifications (PTMs) in a single mass spectrum. Combined with immunoprecipitation, Nuc-MS quantified nucleosome co-occupancy of histo

processed data file

MM379_HEK293T_H2AZ_CST.bw
MM380_HEK293T_H2AZ_ABCAM.bw
MM381_HEK293T_H3_3_EMDMillipore.bw
MM383_HEK293T_H4K16ac_CST.bw
MM384_HEK293T_H3K79me2_CST.bw
MM391_HEK293T_Input.bw

thermocycler. Post amplification libraries were size selected at 250-450bp in length using Agencourt AMPure XP beads from Beckman Cou
ombined with immunoprecipitation, Nuc-MS quantified nucleosome co-occupancy of histone H3.3 with variant H2A.Z (sixfold over bulk) and

processed data file

MM379_HEK293T_XL_H2AZ_CST_peaks.narrowPeak
MM380_HEK293T_XL_H2AZ_ABCAM_peaks.narrowPeak
MM381_HEK293T_XL_H3_3_EMDMillipore_peaks.narrowPeak
MM383_HEK293T_XL_H4K16ac_CST_peaks.narrowPeak
MM384_HEK293T_XL_H3K79me2_CST_peaks.narrowPeak

e selected at 250-450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent Hig
ccupancy of histone H3.3 with variant H2A.Z (sixfold over bulk) and the co-occurrence of oncogenic H3.3K27M with euchromatic marks (for

raw file
MM379_HEK293T_H2AZ_CST_S9_L001_R1_001.fastq.gz
MM380_HEK293T_H2AZ_ABCAM_S10_L001_R1_001.fastq.gz
MM381_HEK293T_H3_3_EMDMillipore_S11_L001_R1_001.fastq.gz
MM383_HEK293T_H4K16ac_CST_S13_L001_R1_001.fastq.gz
MM384_HEK293T_H3K79me2_CST_S14_L001_R1_001.fastq.gz
MM391_HEK293T_Input_S15_L001_R1_001.fastq.gz

om Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit.
currence of oncogenic H3.3K27M with euchromatic marks (for example, a >15-fold enrichment of dimethylated H3K79me2). Nuc-MS is high

raw file raw file raw file

MM379_HEK293T_H2AZ_CST_S9_L002_R1_001.fastq.gz
MM380_HEK293T_H2AZ_ABCAM_S10_L002_R1_001.fastq.gz
MM381_HEK293T_H3_3_EMDMillipore_S11_L002_R1_001.fastq.gz
MM383_HEK293T_H4K16ac_CST_S13_L002_R1_001.fastq.gz
MM384_HEK293T_H3K79me2_CST_S14_L002_R1_001.fastq.gz
MM391_HEK293T_Input_S15_L002_R1_001.fastq.gz

ty DNA Kit.
ethylated H3K79me2). Nuc-MS is highly concordant with chromatin immunoprecipitation-sequencing (ChIP-seq) and offers a new readout o
matin immunoprecipitation-sequencing (ChIP-seq) and offers a new readout of nucleosome-level biology.