Cut tag it assay kit

ChIP-Seq libraries were constructed from FACS-enriched RGCs (100,000 cells for each library) using rabbit anti-H3K27me3 (1:50, Active Motif 39155) or normal rabbit IgG (1:50, Sigma-Aldrich NI01) and the CUTTag-IT Assay Kit (Active Motif) following the manufacture’s manual. Identical amounts of DNA from each library were used in qPCR to determine the enrichment of H3K23me3 in the promoter region of each gene. Two pairs of primers were designed for each gene. One pair for Lingo3 promoter region resulted in no amplification and was excluded from data analysis. Sequences of primers used in qPCR are in Supplemental Table 5. Positive control (71020) and negative control primers (71013) were purchased from Active Motif. Fold enrichment of H3K27me3 binding was determined using the ddCt method and normalized to IgG. All qPCR experiments were done in triplicate.

CUT&Tag was performed with CUT&Tag-IT Assay Kit (53160, ACTIVE MOTIF) in 1.5 × 10⁶ FaDu cells using anti-SF3B2 (sc-514976, Santa Cruz, 5 µL, 1:20 dilution) and anti-H3 (ab1791, Abcam, 1 µL, 1:100 dilution) antibodies. Rabbit anti-mouse IgG (ab6709, Abcam, 1 µL) was used to enhance the signal. The cells were collected using a cell scraper.

HCC827 cells were treated with DMSO (control) or AZD9291 (100 nM) for 24 h. Next, the cells were collected and analyzed using the CUT&Tag-IT Assay Kit (Active Motif, 53160) and histone H3K27ac antibody (Active Motif, 39034), following the manufacturer’s instructions. Genomic libraries containing fragments enriched by binding to anti-histone H3K27ac Ab were sequenced using an Illumina NovaSeq 6000 (Illumina, Inc.). The fastq files were uploaded to Basepair (https://activemotif.basepairtech.com/) and the differences in histone H3K27ac peaks between DMSO and AZD9291 treated samples were searched.

CUT&Tag libraries (two biological replicates) were prepared using the CUT&Tag-IT^™ Assay Kit (Active Motif, 53160) following to the manufacturer’s protocol. 10⁶ of Mel cells were collected for each biological replicate and two replicates were prepared. The Mel cells were bound to Concanavalin A Beads and Incubated with 1:50 rabbit polyclonal Phospho-Rpb1 CTD Ser5 and Ser2 antibody (Cellsignaling 13523 and 13499). Guinea pig a-rabbit antibody was used at 1:100 dilution as secondary antibody. Tagmentation was performed using pA-Tn5 Transposomes at 37°C for 60 mins. DNA was purified by DNA Purification Column, then universal i5 primers and uniquely barcoded i7 primers were added to the DNA with 14cycles of PCR. Individual libraries were purified with SPRI beads and eluted with 20 µL DNA Purification Buffer. The libraries were sequenced on a MiSeq. For data analysis, see Supplementary Materials.

CUT&Tag libraries were prepared using the CUT&Tag-IT Assay Kit (Active Motif, 53160) following to the manufacturer’s protocol. 10⁶ of MEL cells were collected for each biological replicate and two replicates were prepared. The MEL cells were bound to Concanavalin A Beads and Incubated with 1:50 rabbit polyclonal Phospho-Rpb1 CTD Ser5 and Ser2 antibody (Cell Signaling 13523 and 13499). Guinea pig a-rabbit antibody was used at 1:100 dilution as secondary antibody. Tagmentation was performed using pA-Tn5 Transposomes at 37°C for 60 minutes. DNA was purified by DNA Purification Column, then universal i5 primers and uniquely barcoded i7 primers were added to the DNA with 14 cycles of PCR. Individual libraries were purified with SPRI beads and eluted with 20 μL DNA Purification Buffer. The libraries were sequenced on a MiSeq. Antibodies are listed in Table S1.

A total of 100,000 cardiomyocytes isolated from 2-month-old hearts were processed using the CUT&Tag-IT Assay Kit (Active Motif) with the following specifications or modifications: (1) primary antibodies were incubated overnight, (2) the secondary antibody was incubated for 2 h, (3) binding of the pA-Tn5 transposomes with the secondary antibodies was carried out for 2 h and (4) PCR amplification was carried out for 18 cycles. The following antibodies were used: H3K36me2 (Active Motif, 39255; Cell Signaling Technology, 2901; 1 μl of each antibody were combined for each reaction).