H3K4me3 ChIP-seq of hsa-miR-96-5p Overexpressing Cells

Description

H3K4me3 ChIP-seq of hsa-miR-96-5p Overexpressing IMR-90 Human Lung Fibroblasts. Compressed FASTQ archives of Control (C2 and C3) and miR-96-5p overexpressing IMR-90 cells (M2 and M3), forward (R1) and reverse (R2) strands. METHOD. Chromatin Immunoprecipitation and Analysis. Cell cultures for each condition were grown in T-175 flasks, treated as indicated, and processed according to instructions for ChIP-PCR workflows (SimpleChIP® Plus Enzymatic Chromatin IP Kit Agarose Beads #9004) and ChIP-seq workflows (SimpleChIP® Plus Enzymatic Chromatin IP Kit Magnetic Beads #9005). ChIP-PCR primers sequences (5’→3’) are: p16 prom 1 up-TCCTAGTTGTGAGAGCCCCA; p16 prom 1 dn-CCGCGCTCCTGAAAATCAAG; p16 prom 2 up-CAAATCCTCTGGAGGGACCG; p16 prom 2 dn-TGCCACATTCGCTAAGTGCT; p16 prom 3 up-TCGAAGCGCTACCTGATTCC; p16 prom 3 dn-ATCCAGGTGGGTAGAGGGTC; p21 prom 1 up-CTCCATCCCTATGCTGCCTG; p21 prom 1 dn-TTGGGACATGTTCCTGACGG; p21 prom 2 up-ATACGGGCTATGTGGGGAGT; p21 prom 2 dn-TGGTTGCAGCAGCTTTGTTG; p21 prom 3 up-CGTTCACAGGTGTTTCTGCG; p21 prom 3 dn-CACATCCCGACTCTCGTCAC; p21 prom 4 up-CTCTCCGAAAGCTACAGGGC; p21 prom 4 dn-TTTTCCTCACGAAGAGGGCG; p21 prom 5 up-CCGAGATCAACCCCTATGCC; p21 prom 5 dn-TTGGCCTTCCTGCCTTAGAC; IL1b prom 1 up-GAGGGTGTGGGTCTCTACCT; IL1b prom 1 dn-CCCCAGCCAAGAAAGGTCAA; IL1b prom 2 up-CATCCAGTGCATGGCAGTCA; IL1b prom 2 dn-CCTCTGCTCCAGCTCTCCTA; IL1b prom 3 up-GAGGCTAGGGGAGGCTATCA; IL1b prom 3 dn-GCTAGGTCAGTTGTGCAGGT; IL1b prom 4 up-TTTGGCTACTGTGGCACGAT; IL1b prom 4 dn-ATTTTACCGCCCAGCAGGAA;IL6 prom 1 up-AGGATTCCCAAGGGGTCACT; IL6 prom 1 dn-GTCTCCAGGTGGAGTGTGTG; IL6 prom 2 up-TAGATTTGAGGCCAACGGGG; IL6 prom 2 dn-GGAACTTCCTGACACCAGCA; IL6 prom 3 up-ACCAACTTGTCGCACTCACT; IL6 prom 3 dn-ACCCACTTTTTGTTGCTGCC. Immunoprecipitations were done with Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733 (Cell Signaling) and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 (Cell Signaling). For ChIP-PCR immunoprecipitations were done with ChIP-seq libraries were sequenced with ThruPLEX® DNA-Seq Kit (Takara Bio) and sequenced the pooled libraries on a NextSeq 2K P3 2x100-bp run, generating > 1 billion reads. Output FASTQ files were processed with FastQC; Genome Analysis Toolkit (GATK; Broad Institute) was used clean FASTQ files which were then aligned to hg38 with (Burrows-Wheeler Aligner) BWA. The Galaxy Platform (Galaxy Community, 2022) was used for narrow and broad peak calling (MACS2) and differential peak calling (DiffBind).

Steps to reproduce

Chromatin Immunoprecipitation and Analysis. Cell cultures for each condition were grown in T-175 flasks, treated as indicated, and processed according to instructions for ChIP-PCR workflows (SimpleChIP® Plus Enzymatic Chromatin IP Kit Agarose Beads #9004) and ChIP-seq workflows (SimpleChIP® Plus Enzymatic Chromatin IP Kit Magnetic Beads #9005). ChIP-PCR primers sequences (5’→3’) are: p16 prom 1 up-TCCTAGTTGTGAGAGCCCCA; p16 prom 1 dn-CCGCGCTCCTGAAAATCAAG; p16 prom 2 up-CAAATCCTCTGGAGGGACCG; p16 prom 2 dn-TGCCACATTCGCTAAGTGCT; p16 prom 3 up-TCGAAGCGCTACCTGATTCC; p16 prom 3 dn-ATCCAGGTGGGTAGAGGGTC; p21 prom 1 up-CTCCATCCCTATGCTGCCTG; p21 prom 1 dn-TTGGGACATGTTCCTGACGG; p21 prom 2 up-ATACGGGCTATGTGGGGAGT; p21 prom 2 dn-TGGTTGCAGCAGCTTTGTTG; p21 prom 3 up-CGTTCACAGGTGTTTCTGCG; p21 prom 3 dn-CACATCCCGACTCTCGTCAC; p21 prom 4 up-CTCTCCGAAAGCTACAGGGC; p21 prom 4 dn-TTTTCCTCACGAAGAGGGCG; p21 prom 5 up-CCGAGATCAACCCCTATGCC; p21 prom 5 dn-TTGGCCTTCCTGCCTTAGAC; IL1b prom 1 up-GAGGGTGTGGGTCTCTACCT; IL1b prom 1 dn-CCCCAGCCAAGAAAGGTCAA; IL1b prom 2 up-CATCCAGTGCATGGCAGTCA; IL1b prom 2 dn-CCTCTGCTCCAGCTCTCCTA; IL1b prom 3 up-GAGGCTAGGGGAGGCTATCA; IL1b prom 3 dn-GCTAGGTCAGTTGTGCAGGT; IL1b prom 4 up-TTTGGCTACTGTGGCACGAT; IL1b prom 4 dn-ATTTTACCGCCCAGCAGGAA;IL6 prom 1 up-AGGATTCCCAAGGGGTCACT; IL6 prom 1 dn-GTCTCCAGGTGGAGTGTGTG; IL6 prom 2 up-TAGATTTGAGGCCAACGGGG; IL6 prom 2 dn-GGAACTTCCTGACACCAGCA; IL6 prom 3 up-ACCAACTTGTCGCACTCACT; IL6 prom 3 dn-ACCCACTTTTTGTTGCTGCC. Immunoprecipitations were done with Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733 (Cell Signaling) and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 (Cell Signaling). For ChIP-PCR immunoprecipitations were done with ChIP-seq libraries were sequenced with ThruPLEX® DNA-Seq Kit (Takara Bio) and sequenced the pooled libraries on a NextSeq 2K P3 2x100-bp run, generating > 1 billion reads. Output FASTQ files were processed with FastQC; Genome Analysis Toolkit (GATK; Broad Institute) was used clean FASTQ files which were then aligned to hg38 with (Burrows-Wheeler Aligner) BWA. The Galaxy Platform (Galaxy Community, 2022) was used for narrow and broad peak calling (MACS2) and differential peak calling (DiffBind).

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