title: A_9_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 9 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_7_normal_IP

status: Public on Jul 31 2025

type: SRA

city: Guangzhou

first: Jian

last: Zhen

address: 651 Dongfeng East Road

department: State Key Laboratory of Oncology in South China

country: China

institute: Sun Yat-sen University Cancer Center

description: IP sample of MeRIP-seq

library_source: transcriptomic

data_processing:
Reads were first mapped to the human reference genome (GRCh38) by BWA (v0.7.15) and the annotation file obtained from the GENCODE database (https://www.gencodegenes.org/). Circm6A was applied to detect the circRNAs and their m6A modifications from all our PDAC tissue and normal tissue samples.
Genome_build: GRCh38
Supplementary_files_format_and_content: raw read count files of circRNAs for each sample

molecule: total RNA

organism: Homo sapiens

source_name: pancreas tissue

extract_protocol:
Total RNA from pancreatic cancer 7 normal tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.

tag: tissue

value: tumor adjant

tag: disease

value: pancreatic cancer


title: A_9_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 9 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.

value: tumor


title: ZNF274KO_S_Rep4

city: Lausanne

first: Julien

last: Duc

state: VAUD

address: Station 19 CH-1015 Lausanne

department: School of Life Science

country: Switzerland

institute: EPFL

description: RNAseq on ZNF274KO HEK293T  FACS-Sorted in S/G2

data_processing:
Raw reads were mapped with hisat2 with -k5 optino to the human hg19 genome
Gene and transposable elements summaziation performed with featureCounts using -Q 10 as option
Assembly: hg19
Supplementary files format and content: countTable raw unique read counts

growth_protocol: HEK293T were grown in DMEM supplemented with FCS and PSG

extract_protocol:
NucleoSpin RNA Plus, Mini kit for RNA purification with DNA removal column MACHEREY-NAGEL Item number: 740984.50
Illumina stranded mRNA
We performed a "Illumina stranded mRNA ligation" (ISML) prep starting from 200 ng of RNA (see table below), according to Illumina protocol 1000000124518 v03 (internal exp code LPX760). We quantified the libraries by qubit DNA HS and performed profile analysis by TapeStation TS4200. All libraries look good and have sufficient yields.

treatment_protocol: Cells were fixed according to Channathodiyil and Houseley, 2021 stained with DAPI and PE anti-Cyclin A Antibody AB_2715836 (BioLegend Cat. No. 644004) and FACS sorted in eight populations covering the cell cycle

tag: cell line

tag: cell type

value: Fibroblast

tag: genotype

tag: treatment

value: FACS-Sorted in S/G2


title: A_7_normal_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 7 normal tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: ZNF274KO_S_Rep3


title: ZNF274KO_S_Rep2


title: A_6_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 6 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: WT_G2

library_source: genomic

data_processing:
Raw reads mapped with bowtie2 using --sensitive-local parmeter
Bigwigs were computed using bamCoverage from deeptools with parameters  --normalizeUsing CPM --scaleFactor 100 --binSize 50000 --smoothLength 250000
Assembly: hg19
Supplementary files format and content: Bigwigs files containing normliazed counts (CPM)

molecule: genomic DNA

extract_protocol:
According to protocol from Zhao et al ., 2020
BrdU-Seq (NEBNext UltraII DNA Library Prep for Illumina)
NEBNext Ultra DNA Library Prep Kit for Illumina (NEB, cat. no. E7370)

treatment_protocol: No

tag: chip antibody

value: D™ Purified Mouse Anti-BrdU Ref :347580


title: A_6_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 6 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: ZNF274KO_S_Rep1


title: A_58_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 58 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_8_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 8 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: ZNF274KO_G1_Rep4

description: RNAseq on ZNF274KO HEK293T  FACS-Sorted in G1

value: FACS-Sorted in G1


title: A_58_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 58 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: ZNF274KO_G1_Rep2


title: A_57_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 57 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: ZNF274KO_G1_Rep1


title: ZNF274KO_G1_Rep3


title: A_56_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 56 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_55_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 55 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: WT_S_Rep4

description: RNAseq on WT HEK293T  FACS-Sorted in S/G2

value: WT


title: WT_S_Rep3


title: A_57_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 57 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: WT_S_Rep2


title: A_55_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 55 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_55_normal_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 55 normal tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_55_normal_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 55 normal tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: WT_G1_Rep3

description: RNAseq on WT HEK293T  FACS-Sorted in G1


title: WT_G1_Rep4

description: RNAseq on WT HEK293T  FACS-Sorted in G1


title: WT_G1_Rep2

description: RNAseq on WT HEK293T  FACS-Sorted in G1


title: WT_S_Rep1


title: A_53_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 53 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_56_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 56 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_54_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 54 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: ZNF519_D_Rep3

description: RNAseq on K562 overexpressing ZNF519 upon DOX _ treated with DOX for 5 days

growth_protocol: K562 were grown in RPMI supplemented with FCS and PSG

extract_protocol:
NucleoSpin RNA Plus, Mini kit for RNA purification with DNA removal column MACHEREY-NAGEL Item number: 740984.50
Illumina stranded mRNA
We performed a "Illumina stranded mRNA ligation" (ISML) prep starting from 200 ng of RNA (see table below), according to Illumina protocol 1000000124518 v03 (internal exp code LPX760). We quantified the libraries by qubit DNA HS and performed profile analysis by TapeStation TS4200. All libraries look good and have sufficient yields.

treatment_protocol: Cells were treated with DOX during 5 Days

value: Myeloid

value: DOX


title: A_54_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 54 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_53_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 53 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: ZNF519_D_Rep1


title: A_52_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 52 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_52_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 52 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: GFP_D_Rep3

description: RNAseq on K562 overexpressing GFP upon DOX _ treated with DOX for 5 days


title: ZNF519_D_Rep2


title: A_51_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 51 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: GFP_D_Rep2


title: GFP_D_Rep1


title: v9_D7_Rep3

description: RNAseq on K562 treated with shZNF519v9 for 7 days

treatment_protocol: Cells were transduced with LV against ZNF519 or scrambled sequence for 7 days


title: SCR_D7_Rep2

value: shSCR


title: A_50_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 50 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_50_normal_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 50 normal tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: WT_G1_Rep1

description: RNAseq on WT HEK293T  FACS-Sorted in G1


title: quail embryonic stem cells rep2, p11

city: Pasadena

first: Xi

last: Chen

state: California

address: 1200 E. California Blvd

country: USA

institute: California Institute of Technology

description: Library name: sample2

data_processing:
STAR alignment, RSEM quantification, DESeq for differential expression
Assembly: quail reference genome (Ensembl Coturnix_japonica.Coturnix_japonica_2.0.113), goose reference genome (Ensembl Anser_cygnoides.GooseV1.0.113), Ostrich reference genome (Ensembl Struthio_camelus_australis.ASM69896v1.113)
Supplementary files format and content: txt format, including raw counts, gene length, etc

organism: Coturnix japonica

source_name: freshly laid EGK.X embryo

growth_protocol: Freshly laid avian fertile eggs at EGK.X-XII stages were kept at 16 °C before extraction. To harvest cells from yolk, a filter paper-based method was applied (Chapman et al., 2001). Filter paper with a hole at center was placed on top of the embryo. The outer edge of the filter paper was cut together with the vitelline membranes. Embryo were then released from yolk together with the filter paper and washed in PBS solution. Next, embryo was transferred into a 15ml tube free of the paper ring and dissociated by pipetting inside N2B27 medium. Dissociated cells were cultured in tissue plates coated with irradiated mouse embryonic fibroblast (MEF) cells as feeder layer. Basal medium used for routine ESC maintenance was N2B27 medium composed of DMEM/F12:Neuralbasal (1:1) plus 0.5x N2, 0.5x B27 supplement and 20ng/ml ovotransferrin with the addition of 1x penicillin-streptomycin. Avian ESC self-renewal was kept with 2µM IWR-1, 2.5µM Gö6983, 2µM SB431542. Quail and goose ESCs were treated with additional chicken LIF at 10ng/ml. ESCs were cultured at 38 °C in 5% CO2 with daily medium change. To passage, TrypLE was used to incubate the cells for 5 min and neutralized with basal medium in triple amount. Cells were handled with 5ml pipette all the time to avoid dissociation into singles.

extract_protocol:
Total RNA of in-vitro expanded cells was extracted using the RNeasy Mini Kit (QIAGEN) according to manufacturer’s instructions.
RNA-seq

value: freshly laid EGK.X embryo

value: qES p11

value: embryonic stem cells


title: quail embryonic stem cells rep1, p11

description: Library name: sample1


title: A_5_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 5 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_50_normal_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 50 normal tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: v9_D7_Rep2

description: RNAseq on K562 treated with shSCR for 7 days


title: SCR_D7_Rep3


title: A_51_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 51 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_5_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 5 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: RF054_M_3

description: RNAseq on K562 FACS-Sorted in M

data_processing:
Raw reads were mapped with hisat2 with -k5 optino to the human hg19 genome
Gene and transposable emelents summaziation performed with featureCounts using -Q 10 as option
Assembly: hg19
Supplementary files format and content: countTable raw unique read counts

extract_protocol:
NucleoSpin RNA Plus, Mini kit for RNA purification with DNA removal column MACHEREY-NAGEL Item number: 740984.50
NEBNext Ultra II Directional RNA Library Prep with ribodepletion
We performed a NEBNext Ultra II Directional RNA Library Prep with ribodepletion starting from 51 ng of RNAs (see table below), according to NEB protocol NEBNext Ultra II Directional RNA Library Prep Kit, Version 4.0_4/21 (internal exp code PHX056). We quantified the resulting libraries by qubit DNA HS and performed profile analysis by TS4200. All libraries had sufficient yields (see table below), and all looked very good apart from RF053_S3_2 and RF054_M_3, which were still OK but with suboptimal characteristics (see comments in the table below).

value: FACS-Sorted in M


title: SCR_D7_Rep1


title: A_50_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer 50 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_48_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 48 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: RF054_G2_3

description: RNAseq on K562 FACS-Sorted in G2

value: FACS-Sorted in G2


title: A_49_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 49 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_49_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 49 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_48_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 48 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: RF054_S3-G2_3

description: RNAseq on K562 FACS-Sorted in S3-G2

value: FACS-Sorted in S3-G2


title: RF054_S3_3

description: RNAseq on K562 FACS-Sorted in S3

value: FACS-Sorted in S3


title: RF054_S2_3

description: RNAseq on K562 FACS-Sorted in S2

value: FACS-Sorted in S2


title: A_47_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 47 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.

value: tumor

value: pancreatic cancer


title: A_47_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 47 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_46_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 46 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: v9_D7_Rep1

extract_protocol:
NucleoSpin RNA Plus, Mini kit for RNA purification with DNA removal column MACHEREY-NAGEL Item number: 740984.50
Illumina stranded mRNA
We performed a "Illumina stranded mRNA ligation" (ISML) prep starting from 200 ng of RNA (see table below), according to Illumina protocol 1000000124518 v03 (internal exp code LPX760). We quantified the libraries by qubit DNA HS and performed profile analysis by TapeStation TS4200. All libraries look good and have sufficient yields.


title: A_46_normal_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 46 normal tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.

value: tumor adjant


title: RF054_G1-S_3

description: RNAseq on K562 FACS-Sorted in G1-S

value: FACS-Sorted in G1-S


title: RF054_G1_3

description: RNAseq on K562 FACS-Sorted in G1

value: FACS-Sorted in G1


title: RF053_M_2

description: RNAseq on K562 FACS-Sorted in M

value: WT


title: A_46_normal_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 46 normal tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: RF053_G2_2

description: RNAseq on K562 FACS-Sorted in G2

value: Myeloid


title: A_45_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 45 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_45_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 45 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: RF053_S2_2

description: RNAseq on K562 FACS-Sorted in S2


title: RF053_S3-G2_2

description: RNAseq on K562 FACS-Sorted in S3-G2


title: A_45_normal_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 45 normal tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_45_normal_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 45 normal tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: RF048_M_1

description: RNAseq on K562 FACS-Sorted in M


title: RF053_S1_2

description: RNAseq on K562 FACS-Sorted in S1

value: FACS-Sorted in S1


title: RF054_S1_3

description: RNAseq on K562 FACS-Sorted in S1


title: A_42_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 42 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: RF053_G1_2

description: RNAseq on K562 FACS-Sorted in G1

extract_protocol:
NucleoSpin RNA Plus, Mini kit for RNA purification with DNA removal column MACHEREY-NAGEL Item number: 740984.50
NEBNext Ultra II Directional RNA Library Prep with ribodepletion
We performed a NEBNext Ultra II Directional RNA Library Prep with ribodepletion starting from 51 ng of RNAs (see table below), according to NEB protocol NEBNext Ultra II Directional RNA Library Prep Kit, Version 4.0_4/21 (internal exp code PHX056). We quantified the resulting libraries by qubit DNA HS and performed profile analysis by TS4200. All libraries had sufficient yields (see table below), and all looked very good apart from RF053_S3_2 and RF054_M_3, which were still OK but with suboptimal characteristics (see comments in the table below).


title: A_43_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 43 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_46_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 46 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_44_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 44 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: RF048_S2_1

description: RNAseq on K562 FACS-Sorted in S2


title: A_42_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 42 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: RF053_G1-S_2

description: RNAseq on K562 FACS-Sorted in G1-S


title: A_43_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 43 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_42_normal_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 42 normal tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_41_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 41 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: RF053_S3_2

description: RNAseq on K562 FACS-Sorted in S3


title: RF048_S1_1

description: RNAseq on K562 FACS-Sorted in S1


title: RF048_S3-G2_1

description: RNAseq on K562 FACS-Sorted in S3-G2


title: RF048_G1-S_1

description: RNAseq on K562 FACS-Sorted in G1-S


title: RF048_G1_1

description: RNAseq on K562 FACS-Sorted in G1


title: RF048_G2_1

description: RNAseq on K562 FACS-Sorted in G2


title: RF048_S3_1

description: RNAseq on K562 FACS-Sorted in S3


title: A_41_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 41 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_41_normal_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 41 normal tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: RR25_BASELINE_0h

city: Milan

first: Edoardo

last: Pedrini

state: Lombardy

address: Via Olgettina 60

department: Division of Neuroscience

country: Italy

institute: Hospital San Raffaele

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v6.1.2 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: GRCh38
Supplementary files format and content: normCount_GEOsubmission.rds rds object containing the normalized reads as sparse matrix
Supplementary files format and content: metadata_GEOsubmission.tsv Tab-separated values files and matrix files

source_name: Organoids IPS derived

extract_protocol:
For the enzymatic cellular dissociation, 30 organoids, for each experiment and for each donor, were first washed in Hank’s balanced salt solution (HBSS) and then, incubated with a freshly prepared dissociation solution (HBSS, papain and DNAase I)for 45 min at 37°C on an orbital shaker (90 rpm), and gently triturated using a P1000 pipette. Cells were incubated again for 15 min at 37°C on an orbital shaker (90 rpm) centrifuged and gently triturated using a P1000 pipette. After the single cell dissociation, the cell suspension was mixed (1:1) with 10 mg/mL ovomucoid protease inhibitor solution and centrifuged at 300g for 5 min. The pellet was resuspend ed in 1X PBS containing BSA (1:50). Cells were counted using trypan blue and a haemocytometer under bright field. A yield of 700-1,200 cells per μL per sample was targeted. For each condition, cell viability was assessed using Luna II Automated Cell Counter. Samples with more than 80% viable cells were processed for downstream analysis. For droplet-based scRNAseq, libraries were prepared using the Chromium Single Cell 3ʹ v3, according to the manufacturer's protocol (10x Genomics) without modifications, targeting 10,000 cells per sample. Using the Illumina Novaseq platform, all samples were sequenced at 50,000 reads per cells. Preparation of cDNA libraries and sequencing support were performed at the Center for Omics Sciences (San Raffaele Scientific Hospital, Milan).
Library was performed according to the manufacter’s instructions (single cell 3’ v3 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.

value: Organoids IPS derived

value: BASELINE

tag: clone

tag: doxy

tag: exposure

tag: timepoint


title: A_41_normal_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 41 normal tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: RR16_BASELINE_0h

description: 10x Genomics


title: A_44_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 44 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: RR24_BASELINE_0h

description: 10x Genomics


title: A_40_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 40 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: CTR4_BASELINE_0h

description: 10x Genomics


title: A_4_normal_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 4 normal tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_42_normal_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 42 normal tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_39_tumor_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 39 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: mix_CSF.ctrl_24h

description: 10x Genomics

value: mix


title: mix_cytokine_24h

description: 10x Genomics

value: cytokine


title: mix_CSF.MS_24h

description: 10x Genomics


title: A_39_tumor_INPUT

description: Input sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 39 tumor tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.


title: A_39_normal_IP

description: IP sample of MeRIP-seq

extract_protocol:
Total RNA from pancreatic cancer patient 39 normal tissue samples were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP).
Total RNA from  tissue samples of PDAC patients were digested with DNase I and then subjected to two rounds of treatment with RiboMinus (Illumina) to reduce rRNA content.
A total amount of 1.5 μg RNA sample was used as Input. We next used Magna MeRIP m6A Kit (Millipore) for m6A immunoprecipitation (m6A-IP). Briefly, 20 μg of Ribo-off RNA was sheared to about 100 nucleotides in length by metal-ion-induced fragmentation and then purified and incubated with 10 μg anti-m6A antibody (Synaptic Systems, 202003) conjugated beads in 500 μl 1 × immunoprecipitation buffer supplemented with RNase inhibitors at 4 °C overnight. The m6A modified RNA was eluted from the beads with N6-methyladenosine (Sigma-Aldrich) and then purified and resuspended in RNase free H2O. The m6A modified RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction and ethanol precipitation. Sequencing libraries for m6A-IP and Input were prepared and sequenced using Illumina HiSeq2500 SE50 and Illumina HiSeqX Ten PE150, respectively.