GSM5839780: spheroid_ko_3; Mus musculus; RNA-Seq
Spheroid preparation: Approximately 1 cm segment of intestinal tissue was minced with scissors and digested in 2 mg/ml of Collagenase I (Thermo Fisher Scientific) at 37°C for 30-40 min, with vigorous pipetting every 5 min. Solution was filtered through 70 mm cell strainer, and crypts were collected by centrifugation and plated on the 24-well plates in 20 mL of Matrigel (Cornig). Following polymerization of the Matrigel for 10 min at 37°C, 50% L-WRN conditioned medium was added. Spheroids were maintained in the culture in the cycles of three days before dissociation with TrypLE express (Thermo Fisher Scientific) and re-plating. For TAM induced gene ablation, spheroids were cultured for 3 days in 50% L-WRN medium containing 250 nM Z-4-Hydroxytamoxifen (MilliporeSigma). After re-plating, spheroids were cultured in 50% L-WRN without Z-4-Hydroxytamoxifen. Crypt preparation: Approximately 5 cm of small intestinal tissue was dissected, longitudinally opened and extensively washed in PBS. Tissue was placed in 2 mM EDTA in PBS for 30 min on the rotator at 4C. Tissue fragments were placed in 10 mL of  0.1% BCS and pipetted up and down. The pipetting and 0.1% BCS solution exchange was repeated 6 times. Fractions 4-6 were collected and crypts were collected by centrifugation. Caco-2 preparation: shERWOOD UltramiR Lentiviral Inducible shRNA target gene set for gene Fgrf1op was purchased from Transomic technologies. Lentiviral packaging was performed in 293T packaging cell line (ATCC, CRL-3216) with Lipofectamine 2000 (Thermo Fisher Scientific) per manufacturer's protocol. Cells were transfected with psPAX2 packaging plasmid (Addgene No. 11260), pCMV-VSV-G envelope plasmid (Addgene No. 8454) and pZIP-TRE3G-ZsGreen-Puro vector expressing either shRNA targeting Fgfr1op (ULTRA-3199095) or control pZIP-TRE3G-ZsGreen-Puro vector expressing non targeting shRNA (TLNSU4300) per manufacturer instructions. Transduction was conducted with 1 mL of supernatant containing polybrene at final concentration of 8 mg/mL in the 12 well plate, with centrifugation at 900g for 1.5 h. The medium was changed next day. Transfected cells were selected with puromycin added in culture 3 days post transduction. Deletion of Fgfr1op in stably transduced Caco2 cells was induced by adding doxycycline (2 mg/ml) in the cell culture medium. Cells were kept under induction for 3 days, and doxycycline (Sigma) containing medium was changed on day 2. Efficiency of Fgfr1op shRNA induction and Fgfr1op deletion was confirmed by visualizing the expression of ZsGreen reporter with Olympus BX51 microscope and Western blot, respectively Sequencing of RNA isolated from enriched crypts: Total RNA was extracted using RNeasy Micro Kit (Qiagen) per manufacturer's instructions. Samples were prepared according to library kit manufacturer's protocol (SMARTer Low Input RNA Kit, Clontech Takara Bio), indexed, pooled, and sequenced on an Illumina NovaSeq 6000. Base calls and demultiplexing were performed with Illumina's bcl2fastq software and a custom python demultiplexing program with a maximum of one mismatch in the indexing read. RNA-seq reads were then aligned to the Ensembl release 76 primary assembly with STAR version 2.5.1a1. Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 1.4.6-p52. Sequencing of RNA isolated from spheroid lines: Total RNA was extracted using RNeasy Micro Kit (Qiagen) per manufacturer's instructions. Samples were prepared according to library kit manufacturer's protocol (Dynal mRNA Direct Invitrogen) and sequencing was conducted on an Illumina HiSeq. Basecalls and demultiplexing were performed with Illumina's bcl2fastq software and a custom python demultiplexing program with a maximum of one mismatch in the indexing read. Reads were aligned and processed as described for crypt RNA-sequencing.