GSM8580417: GFP_ Setdb1KO, replicate 1, Total RNA-seq; Mus musculus; RNA-Seq
Cell Isolation from the Intestinal Epithelium: To isolate crypts, the ileum was dissected and washed with cold phosphate buffered saline (PBS) using blunt-ended syringe, opened longitudinally with a scissor and cut into 3-5 mm pieces. After extensive washing with cold PBS, the tissue fragments were resuspended in 30 volumes of cold PBS, containing 20 mM EDTA and incubated with constant shaking for 30 min. The tissue fragments allowed to settle by gravity to remove supernatant and resuspended in cold PBS. After vigorous shaking and trituration with 10 ml pipette the tissue pieces were collected by gravity sedimentation. The supernatant was discarded and the PBS extraction was repeated 9 to 12 times, until the supernatant contained mainly crypts, as verified by microscopic examination. The final supernatant containing enriched crypt fraction was passed through 70 m cell strainer and centrifuged at 400g for 5 min. To isolate epithelial cells, the crypt pellets were incubated in 10 volumes of preheated TrypLE Express (Gibco) for 4.5 min at 37 0C. The reactions were stopped by the addition of equal volume of Defined Trypsin Inhibitor (Gibco). The cells were passed through 40 m cell strainer, centrifuged at 400g for 5 min and resuspended in ice-cold PBS containing 1 mM EDTA and 2% FBS. GFP+ cells were sorted using BD FACSAria III cell sorter gated by forward scatter, side scatter and pulse width parameter at a flow rate of 3-5000 events/second.                                                                                                                               Total RNA isolation: Total RNAs were purified from liver tissues by brief homogenization in 10 volumes of Trizol reagent using Polytron device, followed by incubation at room temperature for 5 min and the addition of 0.2 volumes of chloroform and further incubation at room temperature for 3 minutes. The samples were centrifuged at 12000 g for 15 minutes at 40C and the aqueous phase was collected, precipitated by the addition of equal volume of isopropanol. After 10 minutes at room temperature, the RNA was collected by centrifugation at 12000g for 15 minutes. The pellet was resuspended in H2O and re-precipitated with ethanol. The RNA samples were further purified by digestion with 10 units of DNase-I for 10 min at 370C, followed by purification with phenol/chlorophorm extraction and ethanol precipitation.                                                  CUT&Tag assay: CUT&Tag assays were performed as described in (Ref 43). Briefly, nuclei were prepared from the isolated, FACS-sorted GFP+ intestinal epithelial cells by resuspension in 10 volumes of ice-cold Swelling Buffer, containing 10 mM Tris pH 7.5, 2 mM MgCl2, 3 mM CaCl2 and protease inhibitor cocktail (CompleteTM EDTA-free, Roche). After centrifugation at 400g for 5 min at 40C, the pellet was resuspended in Lysis Buffer, containing 10 mM Tris pH 7.5, 2 mM MgCl2, 3 mM CaCl2, 10% glycerol, 1% NP40 and protease inhibitor cocktail. After incubation in ice for 3 min the nuclei were collected by centrifugation for 5 min at 400g, at 40C, washed twice with PBS. Then, 105 nuclei were resuspended in a buffer containing 20 mM HEPES–KOH pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% Glycerol, 0.5 mM Spermidine, protease inhibitor cocktail and incubated for 10 min with Concanavalin A-conjugated magnetic beads (Epicypher) that were previously activated by repeated washings with a buffer containing 20 mM HEPES pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2. The reactions were supplemented with 0.5 volume of a buffer containing 20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, protease inhibitor cocktail, 0.01% Digitonin, 2 mM EDTA and 0.5 g of the anti-H3K9Me3 or IgG negative control primary antibodies. The samples were incubated overnight at 40C by gentle agitation. The beads were then supplemented with a buffer containing 20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, protease inhibitor cocktail, 0.01% Digitonin and 0.5 g of anti-rabbit secondary antibody (Epicypher). After incubation at room temperature for 30 min, the beads were washed twice with the same buffer and resuspended in the same buffer containing 300 mM NaCl. Protein A/G-fused Tn5 transposase protein (CUTANA pA/G-Tn5, Epicypher) was added and the samples were incubated for 1 hour at room temperature followed by washing with a buffer containing 20 mM HEPES-NaOH, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, protease inhibitor cocktail and 0.01% Digitonin. The beads were collected and resuspended in the above buffer containing 10 mM MgCl2, and incubated for 1 hour at room temperature, washed once with 10 mM TAPS pH 8.5 and 0.2 mM EDTA. And incubated with 10 mM TAPS pH 8.5 and 0.1% SDS for 1 hour at 580C, to release DNA. After quenching by the addition of 3 volumes of 0.67% Triton-X100, the samples were used for PCR reaction (14 cycles) using universal i5 primers and barcoded i7 primers. After cleanup with AMPure beads (Beckman Coulter), quantification and quality evaluation in Agilent Bionalyzer, the libraries were sequenced using Illumina NextSeq500 platform Roche). Total RNA-seq libraries were generated using CORALL Total RNA Library preparation kit from Lexogen and sequenced in an Illumina NextSeq 500 system. Single cell RNA-seq libraries were prepared using 10x Genomics Single Cell 3′ Reagent Kit. Single cell ATAC-seq libraries were prepared using Chromium Next GEM Single Cell ATAC Reagent Kit.