Nextseq machine

Total RNA from each sample was quantified by Qubit^® Fluorometer and quality checked by Agilent Bioanalyzer^® RNA 6000 Nano Chip. All samples have high quality RNA with a RIN score > 7. One microgram of total RNA from each sample was used as starting material for paired-end RNA-seq library preparation using NEBNext rRNA Depletion Kit (NEB #E6310) and NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB #E7770) following the manufacturer’s instructions. Libraries were sequenced on an Illumina Next Seq machine (#NextSeq500). Reads were processed using Kallisto and DEGS were called using Sleuth^{41 (link)}.
For single cell RNA-seq analyses, only cells showing at least 5000 detected transcripts were considered. Single-cell experiments were performed as described³⁸ . Briefly, cells were processed using 10× genomics platform (v2.3 kits). Barcodes were demultiplexed using 10× internal pipeline. Expression profiles from MCF7 cells either from red media (n = 1227) or two days of estrogen-deprivation (n = 1193) were then normalized using the R package Scran (v1.6.9)^{42 (link)}. Differential expression between the two conditions was estimated using the Two-sample Likelihood Ratio Test implemented in the LRT function of the MAST R package (v1.4.1)^{43 (link)}.

RNA isolated from distal colonic homogenates (from six Sprouty2^FF and six Sprouty2^IEKO littermates) were barcoded using Illumina index primers and sequenced on a NextSeq machine to obtain single-ended reads of 75 bp length to a depth of ~10 M reads/sample by SeqMatic LLC (San Francisco, CA). FASTQ files were pseudoaligned using kallisto^{76 (link)} to the mouse transcriptome to obtain transcript per million abundance estimates. To compare pathway enrichment, transcript abundances were summed to obtain a per-gene expression value, which was then input into GSEA 4.1.0 (ref. ^{77 (link)}). Enrichment of the KRAS pathway was identified using the Hallmark database of targets^{78 (link)}. Specific analysis for enrichment of ILC2 markers was performed using the GSEA algorithm against a previously identified ILC2 gene set profile enriched in intestinal tissue^{31 (link)} For immune cell estimation in colonic tissue, gene expression data (with HUGO gene symbols) were submitted to the CIBERSORT portal (http://cibersort.stanford.edu) and processed, using the LM22 signature gene file at 100 permutations³⁴ . Results were standardized as relative abundance of the total predicted immune cell populations.

First strand cDNA synthesis for ribosome associated RNA or total RNA was carried out using Superscript IV reverse transcriptase (Invitrogen) with random hexamer priming. The barcode was amplified from cDNA or plasmid using RE_Amp_F and RE_Amp_R primers with Phusion-HF MM (NEB): 98 °C for 1 min, 22 cycles: 98 °C for 10 s, 55 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min. The amplicon was purified using Nucleospin Gel and PCR cleanup kit (Macherey Nagel) and subsequently digested with XhoI and SpeI. The digestion product was purified as before and ligated to the Illumina adapters described in Supplementary Table 1. This product was amplified using Il_Enrich_F and Il_Enrich_R with Phusion HF MM (NEB): 98 °C for 1 min, 21 cycles: 98 °C for 10 s, 66 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min. This product was resolved by agarose gel electrophoresis and the appropriate sized band was excised and purified using Nucleospin Gel and PCR cleanup kit (Macherey Nagel).
The Illumina library was multiplexed and run on four lanes of Illumina NextSeq machine. Barcodes counts were determined. Only barcodes with greater than >10 counts in the cDNA and plasmid pools were used for analysis.

The landing pad cells were seeded as 1 million per well on 6-well plates 24 h before transfection. One µg library plasmid pJC253L (Supplementary Fig. 10) carrying an attB site and the 5′ UTR library and 1 µg BxbI recombinase expressing plasmid pCAG-BxbI were mixed with 6 µL FuGENE HD and added into each well. Eight wells of each landing pad cells were used for transfection. To ensure the reproducibility of our screening results, we maintained >25-fold coverage of each library member throughout the screening pipeline. 4 µg/mL puromycin was added three days post-transfection, and the cells were cultured for at least one more week. The cells were then analyzed using FACS and sorted into three bins based on distinct levels of GFP intensity while the unsorted cells were used as control. We chose three sorting brackets of top 0–2.5%, 2.5–5%, and 5–10%, instead of only one bracket of 0–10%, to reduce the false positive candidates. Only the candidates that were significantly over-represented in all three bins, relative to background (0–100% bin), were selected as candidates for further validation.
For NGS library preparation, the genomic DNA was extracted from each bin and 800 ng were used as the template for PCR amplification with barcoded Pi7 primer. Sequencing was performed at the MIT BioMicro Center facilities on an Illumina NextSeq machine using 150 bp double-end reads.

Sequencing libraries were generated from each backcross pool, using the Illumina Nextera kit. These libraries were sequenced on an Illumina NextSeq machine by the USC Epigenome Center, using 75 × 75 base reads. Rough pools were sequenced to an average of 246-fold and control pools were sequenced to an average of 162-fold coverage. Reads were aligned to either a BY or 3S reference genome, using the Burrows–Wheeler Aligner version 7 with option mem −t 20 (ref. 39 (link)) and mpileup files were generated with SAMtools40 (link). Sequencing data can be accessed from the Sequence Read Archive using Biosample accession numbers SAMN04126845 through SAMN04126912 or the Bioproject accession number PRJNA301897. The specific accession number for each pool is listed in Supplementary Table 6.