Journal: bioRxiv

Article Title: TAF-ChIP: An ultra-low input approach for genome wide chromatin immunoprecipitation assay

doi: 10.1101/299727

Figure Lengend Snippet: (1) Formaldehyde fixed cells were directly sorted into RIPA buffer (see methods for details). (2) Cells were briefly sonicated at low intensity to break open the nuclei. (3) Antibodies were coupled to magnetic beads in the presence of blocking reagents. (4) Antibody coupled beads were added to the cell lysate and incubated overnight at 4°C. (5) The tagmentation reaction was performed after initial washes with low salt IP buffer and homemade tagmentation buffer. (6) The tagmentation reaction and the background regions (not anchored by antibody interaction) were washed away with subsequent high stringency washes. (7) The proteinase K was heat-inactivated and the material was PCR-amplified without purification.

Article Snippet: The washed beads were resupended in 20 μl of 1X tagmentation DNA buffer (Nextera XT Kit) containing 1 μl of Nextera DNA tagmentation enzyme and incubated at 37 °C for 40 min with constant shaking in a thermoblock at 500 rpm.

Techniques: Sonication, Magnetic Beads, Blocking Assay, Incubation, Amplification, Purification

Journal: bioRxiv

Article Title: TAF-ChIP: An ultra-low input approach for genome wide chromatin immunoprecipitation assay

doi: 10.1101/299727

Figure Lengend Snippet: (A) Bioanalyzer profile before and after tagmentation with indicated cell numbers. The amount of Tn5 tagmentase was kept constant in all conditions. (B) Bioanalyzer profile of a representative TAF-ChIP library showing the size distribution of final library fragments. (C) Genome browser track example of H3K27Me3 with TAF-ChIP approach and recently published CUT&RUN method with different cell numbers, as indicated in the labels. The label below the tracks shows the gene model, and the y-axis represents normalized read density in reads per million (rpm). (D) Clusterogram of TAF-ChIP H3K9Me3 (highlighted with a rectangular box) and indicated datasets derived from the signal in the peak file. H3K4Me3 and H3K36Me3 were included as controls. Note that the TAF-ChIP replicates for H3K9Me3 cluster together with the equivalent datasets from ENCODE. The legend in the left indicates the distance based on Jaccard Index. (E) Clusterogram of TAF-ChIP H3K27Me3 datasets (highlighted by a rectangular box) with equivalent datasets from ENCODE, CUT&RUN datasets (from 100, 3000, and 6000 cells), and non-related histone modification datasets used as control. (F) Clusterogram of TAF-ChIP H3K9Me3 datasets (highlighted by a rectangular box) with conventional ChIP-Seq dataset from Drosophila NSCs.

Article Snippet: The washed beads were resupended in 20 μl of 1X tagmentation DNA buffer (Nextera XT Kit) containing 1 μl of Nextera DNA tagmentation enzyme and incubated at 37 °C for 40 min with constant shaking in a thermoblock at 500 rpm.

Techniques: Derivative Assay, Modification, ChIP-sequencing

Journal: bioRxiv

Article Title: In situ 10-cell RNA sequencing in tissue and tumor biopsy samples

doi: 10.1101/444182

Figure Lengend Snippet: Iterative SPRI bead purification eliminates low molecular-weight contaminants before tagmentation. (A) Poly(A) PCR reamplifications of 10-cell human breast cancer samples were analyzed by gel electrophoresis without purification or after one (1x) or two (2x) rounds of purification with 70% (vol/vol) SPRI beads. (B) Contaminating low molecular-weight concatemers are significantly reduced after two rounds of SPRI bead purification. Data are shown as the mean (gray) of n = 3 independent reamplifications (circles) each purified three times (+). Differences were assessed by two-way ANOVA with replication.

Article Snippet: Commercial Tn5 tagmentation kits (Nextera XT) require 1000-fold less material than past microarray hybridizations, prompting reevaluation of how the 10-cell libraries were prepared.

Techniques: Purification, Molecular Weight, Nucleic Acid Electrophoresis