This dataset contains the results of three sequencing experiments: RNAseq, Polysome-seq, m6Aseq. 
A summary of experimental procedures is included below. For full methods details please refer to the associated research article. 
 

RNAseq

Mettl3-flox/flox and VillinCreER;Mettl3-flox/flox ileal enteroids were expanded in Matrigel in 50% WRN media until approximately 8 to 10 million cells per replicate was achieved. 
For each genotype, 3 passage separated replicates from the same enteroid line were used. 
Media was supplemented with 2 uM 4-OHT to induce METTL3 deletion 72 hours prior to collection. 
Libraries were prepared from total RNA using the Smarter® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara Bio 634411) and sequenced on a Novaseq 6000, SP Reagent Kit 1.5 (100 cycles).
Libraries were demultiplexed with Illumina DRAGEN.
The reads were aligned to all transcripts on the mouse reference chromosomes (Gencode version M31, GRCm39) using Kallisto.

Polysome-seq (note that polysome-seq files have "riboseq" prefix")

Mettl3-flox/flox and VillinCreER;Mettl3-flox/flox ileal enteroids were expanded in Matrigel in 50% WRN media until approximately 8 to 10 million cells per replicate was achieved. 
For each genotype, 3 passage separated replicates from the same enteroid line were used. 
Media was supplemented with 2 uM 4-OHT to induce METTL3 deletion 72 hours prior to collection. 
Polysomal RNAs were isolated as described in: 
A. L. Cope, S. Vellappan, J. S. Favate, K. S. Skalenko, S. S. Yadavalli, P. Shah, Exploring Ribosome-Positioning on Translating Transcripts with Ribosome Profiling. Methods Mol. Biol. 2404, 83–110 (2022).
Once the rRNA-depleted polysomal RNA was obtained, it was pooled together, and the libraries were prepared. 
The final library contains a 5' adapter – 4 random bases – insert – 5 random bases – sample barcode – 3’ adapter. 
The randomized bases function as Unique molecular identifiers (UMIs) for deduplication. 
The multiplexed library was then sequenced on Illumina HiSeq 4000 with PE150 runs (paired-end reading of 150 bases), with a sequencing depth of 60 million raw reads per sample. 
For computational analysis, the raw sequencing data were demultiplexed, the adaptors were removed using cutadapt, the contaminant sequences (rRNA and tRNA) were depleted, and the reads were deduplicated (umi_tools dedup). 
The reads were aligned to all transcripts on the mouse reference chromosomes (Gencode version M31, GRCm39) using Kallisto.
Translation efficiency (TE) was calculated by dividing the TPM in the total RNA library by the TPM in the polysome-seq library for each individual transcript and sample.

m6Aseq

Three wildtype C57BL/6J mice (Jax #000664, 2 male 1 female) aged 8 weeks were used. 
Intestinal crypts were isolated from the distal half of the small intestine. 
Isolated crypts were dissociated to single cells, epithelial cells were stained with fluorescent antibodies marking epithelial cells. 
Approximately 800K live epithelial cells per mouse were then isolated by flow cytometry.
RIP-seq was performed according to the “Refined RIP-seq” protocol for low input material.
In brief, 5% of total RNA was set aside as input, remaining RNA was immunoprecipitated twice in succession with two independent m6a-specific antibodies.
Final cDNA Libraries were prepared from twice immunoprecipitated RNA and fragmented input total RNA using the Smarter® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara Bio 634411) and sequenced on a Novaseq 6000, SP Reagent Kit 1.5 (100 cycles). 
Raw reads were aligned to mm10 (gencode_M23_GRCm38.p6) using STAR aligner (2.7.9a). 
N6-methyladenosine peaks were identified using exomePeak2 (version 1.2.0)

Description of the data and file structure

Fastq files are provided. Files are demultiplexed but aligned and unfiltered. The pipeline used for mapping and analysis is described above.

RNAseq

Files with prefix "C" are CTRL mettl3-flox/flox samples
Files with prefix "KO" are METTL3 KO vilcreert2;mettl3-flox/flox samples

Polysomeseq

Files with prefix "Riboseq_CTRL" are CTRL mettl3-flox/flox samples
Files with prefix "Riboseq_METTL3KO" are METTL3 KO vilcreert2;mettl3-flox/flox samples

m6Aseq

Files with prefix ending in "i" are input RNA
Files wih prefix ending in "r" are m6A-RIP RNA
Prefix "M2" and "M3" refer to male mouse replicates
Prefix "F3" refers to female mouse replicates