title: GSM6733475: Cγ1ATG7fl/fl  (Exp), 10d.p.i_scATAC-seq; Mus musculus; ATAC-seq
library_name: GSM6733475
library_construction_protocol: Freshly collected total splenic cells were isolated from spleens of 10- or 21-days immunized mice by tissue homogenization. Cells were resuspended in staining buffer (5% FBS in PBS 1X) and preserved at 4C during all process. Red blood cells were lysed using RBC lysis buffer (Biolegend). Library was performed according to the manufacter's instructions (Chromium Next GEM Single Cell ATAC v1.1, 10x Genomics). Briefly,  Nulcei from splenocytes were obeined after incubation 2 mins in 0.1X lysis buffer.  Nuclei were resuspended in DIluted Nuclei Buffer to get the corresponding Nuclei Stock solution. Nuclei suspensions are incubated in a Transposition Mix that includes a Transposase. Simultaneously, adapter sequences are added to the ends of  the DNA fragments. GEMs are generated by combining  barcoded Gel Beads, transposed  nuclei, a Master Mix, and  Partitioning Oil on a Chromium  Next GEM Chip.  Upon GEM generation, the Gel  Bead is dissolved. Oligonucleotides  containing (i) an Illumina® P5  sequence, (ii) a 16 nt 10x Barcode  and (iii) a Read  1 (Read 1N)  sequence are released and mixed  with DNA fragments and Master  Mix. Thermal cycling of the GEMs  produces 10x barcoded singlestranded DNA. After incubation,  the GEMs are broken and pooled  fractions are recovered.  Solid Phase Reversible Immobilization (SPRI) beads are  used to eliminate unused barcodes from the sample. P7 and a sample index  are added during library  construction via PCR. The  final libraries contain the  P5 and P7 sequences  used in Illumina® bridge  amplification.  corresponding Nuclei Stock Concentrations  corresponding Nuclei Stock Concentrationssplenocytes were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.