GSM5571781: RNA_Ctrl_3_ISC; Mus musculus; RNA-Seq
cell isolation protocol: For colon crypt cell isolation, take colon carefully and put into 10 cm dish containing cold PBS, associated fat tissue was removed carefully. Flush the lumen of the intestine with cold PBS, cut the colon longitudinally with scissors and place in a 50ml tube with 25ml cold PBS, invert 10~15 times and remove PBS (repeat until the supernatant no longer contains any visible debris), cut the colon into around 5mm pieces and place into cold 10ml 5mM EDTA-PBS. Vigorously triturate the fragments by pipetting up and down into a 10ml pipette 15 times, Let the fragments settle by gravity for 30 sec, aspirate the supernatant (Be careful to avoid the intestinal fragments). Replace with 10ml 5mM EDTA-PBS and place at 4°C on roller for 15 min and remove the EDTA-PBS from the tube by aspiration. Wash with PBS twice and add 3ml Basal media (RPMI 1640) containing 500U/ml Collagenase Type IV and pipette up and down 5 times using 5ml pipette. Place the tubes in 37°C water bath for 30-40min. Add 10ml cold PBS and then vigorously vortex with a 10ml pipette (x10 times), Collect this 10 ml supernatant fraction in a separate tube. Repeat the cold PBS/vortex 2 more times, and combined 2nd and 3rd round wash into 50ml tube to gather crypt cells. Omni-ATAC protocol (Corces et al., 2017). Briefly, 50000 cells were washed and lysed in lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2) containing 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin for 10 min on ice to prepare the nuclei. ImmediWe followed the Omni-ATAC protocol (Corces et al., 2017). Briefly, 50000 cells were washed and lysed in lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2) containing 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin for 10 min on ice to prepare the nuclei. Immediately after lysis, nuclei were spun at 500g for 10 min to remove the supernatant. Nuclei were then incubated with the Tn5 transposome and tagmentation buffer (20 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 20% Dimethylformamide) containing 0.1% Tween-20 and 0.005% Digitonin at 37 °C for 30 min. After the tagmentation, the stop buffer was added directly into the reaction to end the tagmentation. ately after lysis, nuclei were spun at 500g for 10 min to remove the supernatant. Nuclei were then incubated with the Tn5 transposome and tagmentation buffer (20 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 20% Dimethylformamide) containing 0.1% Tween-20 and 0.005% Digitonin at 37 °C for 30 min. After the tagmentation, the stop buffer was added directly into the reaction to end the tagmentation. PCR was performed to amplify the library. ATAC-seq: PCR was performed to amplify the library. After the PCR reaction, libraries were purified with SPRI (Beckman) beads and deep sequencing was performed by Sequencing Facility in NCI. For RNA-seq, the mRNA from fresh isolated 2-5 million colon crypt ISCs was isolated by using RNeasy Plus Micro Kit (Qiagen, cat:74034) and the concentration and QC was determined by Agilent technologies. RNA-Seq samples were pooled and sequenced on NextSeq using Illumina TruSeq Stranded mRNA Library Prep and paired-end sequencing. 