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P. Van-de, After digestion cells were washed with RPMI 2% FBS (Sigma-Aldrich F7524), centrifugated (1250rpm at 4 C for 7 min) and resuspended. RORgt eGFP embryos were collected between embryonic days E11.5-E14.5 and 4OHT pulsed iCdh5 tdT or iCxcr4 tdT embryos were collected at E13.5 or E16.5. Fetal livers (FL) were dissected from embryos and isolated for analysis of LTi cells. Brains of fate mapped embryos were isolated for analysis of tdTomato recombination in microglia. Limbs and all internal organs were removed to enrich for peripheral LN anlagen (except for E11.5 RORgt eGFP embryos being too premature to remove all organs) and checked for LTi cells. This tissue is later mentioned as periphery. Tissues were gently digested as described above. Cells were blocked (15% normal mouse serum (Jackson Immunoresearch 015-000-120), 4OHT -Sigma T176) and 0.6mg of progesterone (Sigma P0130) at different embryonic stages (from E7.5 to E10.5). For adult fatemapping experiments, pups were delivered by C section and cross fostered with lactating CD1 females. For lymph node (LN) formation analysis, pp.0-4333, 2014.

. Rankin, Soft tissues including spleens, LNs and bone marrows (after the cells have been flushed from the bones) were gently digested as described previously. Red blood cell lysis was also performed on spleen and bone marrow samples prior to staining. Brains were prepared using the brain dissociation kit from Miltenyi (Miltenyi Biotec 130-107-677). Guts were prepared as described previously, 20mL buffer A (HEPES 1M (GIBCO 15630-80), EDTA (Invitrogen 15576-028) 0.5M, 10% FBS in PBS). EDTA was washed 4 times with PBS and scissor minced pieces were, vol.1, p.10, 0200.

. Fbs and . Collagenase, Remaining aggregates were mechanically disrupted using a syringe and cells were filtered over a 70mm cell strainer and washed with RMPI 10% FBS. After centrifugation (1500rpm at room temperature (RT) for 10 min), cells were resuspended in 5mL 100% Percoll, vol.5, p.40

, Bone marrows were stained for HSC progenitors as described previously. Brain samples were stained for CD45, Percoll. Cells were centrifuged for 20 min without break (2100rpm at RT) and middle ring cells were harvested and washed with RPMI 10% FBS. Spleen, gut and LN samples were stained for CD45, CD11b (BD biosciences 563015), F4/80, CD3e (Ebiosciences 47-0031-80), CD19 (BD biosciences 562956), CD8a (BD biosciences 560776), Ly6G (Biolegend BLE127633), TCRb (BD biosciences 563221), IL7Ra, a 4 b 7 , CD4 and zombie UV. Blood samples were stained for CD45, vol.11, p.115, 135524.

, Sections were first dehydrated in acetone (Carlo Erba chemicals 301505) for 10 min and after rehydratation in PBS, they were blocked (15% normal donkey serum (NDS, Jackson Immunoresearch 017-000-121), 2% BSA (ID bio 1000-70) in PBS) for 15 min and incubated with rat anti-CD4 (clone GK1.5, eBioscience 14-0041-82) antibody diluted in EBSS 1% Triton X-100 2% BSA for 1h at RT. Sections were washed with PBS and incubated with donkey anti-rat Alexa Fluor 488 (ThermoFisher A-21208) for 45 min at RT, washed with PBS and mounted with Mowiol 4-88 (Calbiochem 475904) plus 1,4-diazabicyclo[2.2.2]octane (Sigma-Aldrich D27802) mounting solution. Images were acquired using Zeiss LSM 780 and 880 confocal laser scanning microscopes and analyzed using Fiji software. Whole mount preparation and analysis E11.5, E12.5, E13.5 or E14.5 RORgt eGFP embryos were fixed in 0.4% PFA overnight at 4 C and subsequently placed in blocking medium (PBS-MT (1% skim milk, 0.4% Triton X-100 in PBS), 5% NDS) for three days. Embryos were incubated with anti-GFP (AVES, GFP-1020) and rat anti-CD4 in PBS-MT + 3% NDS for 7 nights and after washing with PBS + 0.2% Triton X-100 incubated with donkey anti-chicken Cy3 (Jackson Immunoresearch 703-166-155) and donkey anti-rat Alexa 647, Immunofluorescence 4OHT pulsed iCdh5 tdT or iCxcr4 tdT embryos were collected at E13.5. Embryos were fixed by immersion in 4% paraformaldehyde (PFA -Electron Microscopy Science, ref 15714) for 30 min at RT, subsequently cryoprotected with 10% sucrose (Sigma-Aldrich S9378) and 30% sucrose in PBS overnight, frozen in OCT and 20mm cryosections were cut

, Cells were subsequently analyzed by Flow-cytometry. Next generation sequencing For the bulk RNA sequencing, RORgt eGFP E13.5 embryos were isolated and both FL or the embryonic periphery, enriched for peripheral LN anlagen and thus without thymus, gut or other organs were dissociated using the above-mentioned protocol. CD45 + cells were enriched using CD45 magnetic microbeads (Miltenyi Biotec 130052301) on the autoMACS Pro separator. A lineage cocktail was used for removing possible T, B, dendritic cells and macrophages (CD3e (BD biosciences 562286, CD19 (BD biosciences 562291), CD8a (BD biosciences 562283), Ly6G (BD biosciences 562700), F4/80 (BD biosciences 565613)). Cells were subsequently stained and the common lymphoid progenitor (CLP) (alive Lin -CD45 + IL7Ra + a 4 b, vol.7

+. Rorgt and -. , LTi 0 (alive Lin -CD45

, NextSeq 500 data were demuLTiPlexed individually per sample. Furthermore, the raw fastq files were processed to trim the molecular barcodes, the end of reads depending on quality, and the potential remaining illumina adapters. A sequencing Quality Control (QC) step was run twice on the fastq file: before and after the trimming of the reads. Afterward, alignments were performed using STAR versus GRCm38 ensemble transcriptome resulting in a BAM which was qualified by Qualimap RNaseq. Detection and elimination of duplicated reads were done using MarkDuplicates. Finally, one table count by run was obtained using FeatureCount. Bioinformatics analysis were done in R using house made scripts. Normalization of counts and detection of differentially expressed genes was done using DEseq2 R package, PCA was done using the ade4 R package. For the single cell sequencing C57BL/6J E13.5 and E14.5 embryonic peripheries and FL were collected and cell suspension were prepared as mentioned above. CD45 + cells were enriched using CD45 magnetic microbeads on the autoMACS Pro separator. Cells were subsequently stained and alive CD45 + F4/80 -IL7Ra + LTi cells were sort-purified using the BD FACS ARIA III sorter. Sorted-purified cells were subsequently prepared for single-cell sequencing using the Chromium Single Cell 3 0 kit from 10X genomics (v2 for periphery and v3 for FL (120267 and 1000092 respectively-California, USA) for the library preparation and the NextSeq 500/550 High Output Kit v2 (75 cycles) cartridges from Illumina for the sequencing. The quality and concentration of the cDNA was determined with the bioanalyzer using the High Sensitivity DNA Analysis Kit (Agilent 5067-4626) and Qubit fluorometer using the Qubit dsDNA HS Assay Kit. FASTQ raw files were processed using Cell Ranger software (v3, default parameters), which performs alignment, filtering, barcode counting and unique molecular identifier (UMI) counting. Reads were aligned to the mouse mm10 genome. Quality control and cell filtering: Quality control (QC) was performed to remove poor quality cells thanks to in-house developed R scripts, RORgt + CD4 -) and LTi 4 (alive Lin -CD45 -purified using the BD FACS ARIA III sorter. RNA was isolated using a RNA purification kit (Norgen Biotek Corp. 51800) and the quality and concentration of the RNA was determined with the bioanalyzer using RNA 6000 pico kit (Agilent 5067-1513) and Qubit fluorometer (ThermoFisher) using the Qubit dsDNA HS Assay Kit (ThermoFisher Q32854), vol.32, 2020.

, E12.5, E13.5 or E14.5 RORgt eGFP embryo using IMARIS software (version 9.1.2, Bitplane). CLN, MdLN, AxLN, BrLN, RenLN, IngLN and PopLN anlagen were analyzed, QUANTIFICATION AND STATISTICAL ANALYSIS Quantification of LN formation LN size was analyzed by counting the number of CD4 + and GFP + cells at the right sides of the E11, vol.5